WorldWideScience

Sample records for crystallization preliminary x-ray

  1. Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-β-lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Nandini, E-mail: nandini-sharma@merck.com; Toney, Jeffrey H.; Fitzgerald, Paula M. D.

    2005-02-01

    Crystallization and preliminary X-ray analysis of the CphA metallo-β-lactamase from A. hydrophilia are described. The crystals belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 40.75, b = 42.05, c = 128.88 Å, and diffract to 1.8 Å.

  2. Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum

    International Nuclear Information System (INIS)

    Lyashenko, Andrey V.; Belova, Oksana; Gabdulkhakov, Azat G.; Lashkov, Alexander A.; Lisov, Alexandr V.; Leontievsky, Alexey A.; Mikhailov, Al’bert M.

    2011-01-01

    The purification, crystallization and preliminary X-ray structure analysis of the laccase from G. lucidum are reported. The ligninolytic enzymes of the basidiomycetes play a key role in the global carbon cycle. A characteristic property of these enzymes is their broad substrate specificity, which has led to their use in various biotechnologies, thus stimulating research into the three-dimensional structures of ligninolytic enzymes. This paper presents the purification, crystallization and preliminary X-ray analysis of the laccase from the ligninolytic basidiomycete Ganoderma lucidum

  3. Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris

    International Nuclear Information System (INIS)

    Kallio, Juha Pekka; Jänis, Janne; Nyyssölä, Antti; Hakulinen, Nina; Rouvinen, Juha

    2009-01-01

    The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported. Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution

  4. Crystallization and preliminary X-ray analysis of isomaltase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Yamamoto, Keizo; Miyake, Hideo; Kusunoki, Masami; Osaki, Shigeyoshi

    2008-01-01

    The crystallization and preliminary X-ray analysis of isomaltase is reported. Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 Å, β = 91.17°. X-ray diffraction data were collected to 1.35 Å resolution from a single crystal on a synchrotron-radiation source

  5. Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin.

    Science.gov (United States)

    Jaimohan, S M; Naresh, M D; Arumugam, V; Mandal, A B

    2009-10-01

    Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemoglobins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 A resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 A, beta = 109.35 degrees . Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

  6. Crystallization and preliminary X-ray analysis of Escherichia coli RNase G

    International Nuclear Information System (INIS)

    Fang, Pengfei; Wang, Jing; Li, Xu; Guo, Min; Xing, Li; Cao, Xu; Zhu, Yi; Gao, Yan; Niu, Liwen; Teng, Maikun

    2009-01-01

    Full-length E. coli RNase G was overexpressed, purified and crystallized. Diffraction data were collected to a resolution of 3.4 Å. The homologous RNases RNase E and RNase G are widely distributed in bacteria and function in many important physiological processes, including mRNA degradation, rRNA maturation and so on. In this study, the crystallization and preliminary X-ray analysis of RNase G from Escherichia coli is described. Purified recombinant E. coli RNase G, which has 497 amino acids, was crystallized in the cubic space group F432, with unit-cell parameters a = b = c = 219.84 Å. X-ray diffraction data were collected to a resolution of 3.4 Å

  7. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lu, Feifei; Gao, Feng; Li, Honglin; Gong, Weimin; Zhou, Lin; Bi, Lijun

    2014-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å

  8. Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Feifei [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gao, Feng [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Li, Honglin [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China); Gong, Weimin [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of (China); Zhou, Lin, E-mail: gdtb-bg@vip.163.com [Center for Tuberculosis Control of Guangdong Province, Guangzhou, People’s Republic of (China); Bi, Lijun, E-mail: gdtb-bg@vip.163.com [East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of (China)

    2014-07-23

    The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv3705c from M. tuberculosis are described. The conserved protein Rv3705c from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 198.0, c = 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

  9. Expression, crystallization and preliminary X-ray analysis of the phosphoribosylglycinamide formyltransferase from Streptococcus mutans

    International Nuclear Information System (INIS)

    Zhai, Fangli; Liu, Xiaojuan; Ruan, Jing; Li, Jing; Liu, Zhenlong; Hu, Yulin; Li, Shentao

    2011-01-01

    Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was expressed in E. coli, purified and studied crystallographically. Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS–PAGE and MALDI–TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Å resolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å

  10. Crystallization and preliminary X-ray diffraction analysis of rat autotaxin

    International Nuclear Information System (INIS)

    Day, Jacqueline E.; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Hausmann, Jens; Kamtekar, Satwik

    2010-01-01

    Autotaxin (ATX), a pyrophosphatase/phosphodiesterase enzyme, is a promising drug target for many indications and is only distantly related to enzymes of previously determined structure. Here, the cloning, expression, purification, crystallization and preliminary diffraction analysis of ATX are reported. Rat autotaxin has been cloned, expressed, purified to homogeneity and crystallized via hanging-drop vapour diffusion using PEG 3350 as precipitant and ammonium iodide and sodium thiocyanate as salts. The crystals diffracted to a maximum resolution of 2.05 Å and belonged to space group P1, with unit-cell parameters a = 53.8, b = 63.3, c = 70.5 Å, α = 98.8, β = 106.2, γ = 99.8°. Preliminary X-ray diffraction analysis indicated the presence of one molecule per asymmetric unit, with a solvent content of 47%

  11. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    Energy Technology Data Exchange (ETDEWEB)

    Kiyota, Eduardo [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Sousa, Sylvia Morais de [Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas-SP (Brazil); Santos, Marcelo Leite dos; Costa Lima, Aline da [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil); Menossi, Marcelo [Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Campinas-SP (Brazil); Yunes, José Andrés [Laboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas-SP (Brazil); Aparicio, Ricardo, E-mail: aparicio@iqm.unicamp.br [Laboratório de Biologia Estrutural, Instituto de Química, Universidade Estadual de Campinas, CP 6154, 13083-970 Campinas-SP (Brazil)

    2007-11-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR.

  12. Crystallization and preliminary X-ray diffraction analysis of maize aldose reductase

    International Nuclear Information System (INIS)

    Kiyota, Eduardo; Sousa, Sylvia Morais de; Santos, Marcelo Leite dos; Costa Lima, Aline da; Menossi, Marcelo; Yunes, José Andrés; Aparicio, Ricardo

    2007-01-01

    Preliminary X-ray diffraction studies of apo maize aldose reductase at 2.0 Å resolution are reported. Maize aldose reductase (AR) is a member of the aldo-keto reductase superfamily. In contrast to human AR, maize AR seems to prefer the conversion of sorbitol into glucose. The apoenzyme was crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.2, b = 54.5, c = 100.6 Å and one molecule in the asymmetric unit. Synchrotron X-ray diffraction data were collected and a final resolution limit of 2.0 Å was obtained after data reduction. Phasing was carried out by an automated molecular-replacement procedure and structural refinement is currently in progress. The refined structure is expected to shed light on the functional/enzymatic mechanism and the unusual activities of maize AR

  13. Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin

    International Nuclear Information System (INIS)

    Jaimohan, S. M.; Naresh, M. D.; Arumugam, V.; Mandal, A. B.

    2009-01-01

    Parakeet (Psittacula krameri) haemoglobin has been purified and crystallized under low salt buffered conditions. Preliminary analysis of the crystal that belonged to monoclinic system (C2) is reported. Birds often show efficient oxygen management in order to meet the special demands of their metabolism. However, the structural studies of avian haemoglobins (Hbs) are inadequate for complete understanding of the mechanism involved. Towards this end, purification, crystallization and preliminary X-ray diffraction studies have been carried out for parakeet Hb. Parakeet Hb was crystallized as the met form in low-salt buffered conditions after extracting haemoglobin from crude blood by microcentrifugation and purifying the sample by column chromatography. Good-quality crystals were grown from 10% PEG 3350 and a crystal diffracted to about 2.8 Å resolution. Preliminary diffraction data showed that the Hb crystal belonged to the monoclinic system (space group C2), with unit-cell parameters a = 110.68, b = 64.27, c = 56.40 Å, β = 109.35°. Matthews volume analysis indicated that the crystals contained a half-tetramer in the asymmetric unit

  14. Crystallization and preliminary X-ray diffraction analysis of the middle domain of Paip1

    International Nuclear Information System (INIS)

    Kanaan, Ahmad Seif; Frank, Filipp; Maedler-Kron, Chelsea; Verma, Karan; Sonenberg, Nahum; Nagar, Bhushan

    2009-01-01

    The crystallization of the putative MIF4G domain of Paip1 is described. The crystals belonged to the monoclinic space group P2 1 and diffracted X-rays to beyond 2.2 Å resolution. The poly(A)-binding protein (PABP) simultaneously interacts with the poly(A) tail of mRNAs and the scaffolding protein eIF4G to mediate mRNA circularization, resulting in stimulation of protein translation. PABP is regulated by the PABP-interacting protein Paip1. Paip1 is thought to act as a translational activator in 5′ cap-dependent translation by interacting with PABP and the initiation factors eIF4A and eIF3. Here, the crystallization and preliminary diffraction analysis of the middle domain of Paip1 (Paip1M), which produces crystals that diffract to a resolution of 2.2 Å, are presented

  15. Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima

    International Nuclear Information System (INIS)

    Lyashenko, Andrey V.; Zhukhlistova, Nadegda E.; Gabdoulkhakov, Azat G.; Zhukova, Yuliya N.; Voelter, Wolfang; Zaitsev, Viatcheslav N.; Bento, Isabel; Stepanova, Elena V.; Kachalova, Galina S.; Koroleva, Ol’ga V.; Cherkashyn, Evgeniy A.; Tishkov, Vladimir I.; Lamzin, Victor S.; Schirwitz, Katja; Morgunova, Ekaterina Yu.; Betzel, Christian; Lindley, Peter F.; Mikhailov, Al’bert M.

    2006-01-01

    The crystallization and preliminary X-ray structure at 1.9 Å resolution of the fungal laccase from C. maxima are presented. Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 Å resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 Å (R factor = 18.953%; R free = 23.835; r.m.s.d. bond lengths, 0.06 Å; r.m.s.d. bond angles, 1.07°) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002 ▶) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 Å X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO 2 substituents

  16. Crystallization and preliminary X-ray analysis of alginate importer from Sphingomonas sp. A1

    International Nuclear Information System (INIS)

    Maruyama, Yukie; Itoh, Takafumi; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2012-01-01

    Alginate importer from Sphingomonas sp. A1 is a member of the ABC transporter superfamily that directly transports alginate polysaccharide into the cytoplasm. Crystals of alginate importer in complex with the periplasmic binding protein AlgQ2 diffracted X-rays to 3.3 Å resolution. Sphingomonas sp. A1 directly incorporates alginate polysaccharides through a ‘superchannel’ comprising a pit on the cell surface, alginate-binding proteins in the periplasm and an ABC transporter (alginate importer) in the inner membrane. Alginate importer, consisting of four subunits, AlgM1, AlgM2 and two molecules of AlgS, was crystallized in the presence of the binding protein AlgQ2. Preliminary X-ray analysis showed that the crystal diffracted to 3.3 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 72.5, b = 136.8, c = 273.3 Å, suggesting the presence of one complex in the asymmetric unit

  17. The purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Clostridium botulinum

    International Nuclear Information System (INIS)

    Dobson, Renwick C. J.; Atkinson, Sarah C.; Gorman, Michael A.; Newman, Janet M.; Parker, Michael W.; Perugini, Matthew A.

    2008-01-01

    Dihydrodipicolinate synthase (DHDPS), an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the expression, purification, crystallization and preliminary diffraction analysis of DHDPS from C. botulinum are reported. In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. This enzyme, which is part of the diaminopimelate pathway leading to lysine, couples (S)-aspartate-β-semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from Clostridium botulinum, an important bacterial pathogen, are presented. The enzyme was crystallized in a number of forms, predominantly using PEG precipitants, with the best crystal diffracting to beyond 1.9 Å resolution and displaying P4 2 2 1 2 symmetry. The unit-cell parameters were a = b = 92.9, c = 60.4 Å. The crystal volume per protein weight (V M ) was 2.07 Å 3 Da −1 , with an estimated solvent content of 41%. The structure of the enzyme will help guide the design of novel therapeutics against the C. botulinum pathogen

  18. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

    International Nuclear Information System (INIS)

    Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

    2012-01-01

    Chandipura virus glycoprotein ectodomain (Gth) was purified and crystallized at pH 7.5. X-ray diffraction data set was collected to a resolution of 3.1 Å. Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G th ) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G th obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal

  19. Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase CTD

    International Nuclear Information System (INIS)

    Darmon, Amélie; Piton, Jérémie; Roué, Mélanie; Petrella, Stéphanie; Aubry, Alexandra; Mayer, Claudine

    2012-01-01

    The M. tuberculosis DNA gyrase A C-terminal domain (CTD) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2 1 2 1 2 1 and diffraction data were collected to a resolution of 1.55 Å. Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolone in the treatment of tuberculosis. The C-terminal domain (CTD) of the DNA gyrase A subunit possesses a unique feature, the ability to wrap DNA in a chiral manner, that plays an essential role during the catalytic cycle. A construct of 36 kDa corresponding to this domain has been overproduced, purified and crystallized. Diffraction data were collected to 1.55 Å resolution. Cleavage of the N-terminal His tag was crucial for obtaining crystals. The crystals belonged to space group P2 1 2 1 2 1 , with one molecule in the asymmetric unit and a low solvent content (33%). This is the first report of the crystallization and preliminary X-ray diffraction studies of a DNA gyrase CTD from a species that contains one unique type II topoisomerase

  20. Crystallization and preliminary X-ray analysis of the RAD protein from Antirrhinum majus

    International Nuclear Information System (INIS)

    Stevenson, Clare E. M.; Burton, Nicolas; Costa, Manuela; Nath, Utpal; Dixon, Ray A.; Coen, Enrico S.; Lawson, David M.

    2005-01-01

    An 8 kDa proteolytic fragment of the A. majus RADIALIS protein was crystallized and X-ray data were collected to 2 Å resolution. Crystals of the RADIALIS protein from Antirrhinum majus were grown by vapour diffusion after limited proteolysis. Mass spectrometry indicated that an 8 kDa fragment had been crystallized corresponding to the predicted MYB DNA-binding domain. X-ray data collected at room temperature were consistent with tetragonal symmetry, whereas data collected at 100 K using crystals cryoprotected by supplementing the mother liquor with ethylene glycol conformed to orthorhombic symmetry. It was subsequently shown that crystals soaked in cryoprotectants that were ‘osmolality-matched’ to the mother liquor retained tetragonal symmetry. Using these crystals, X-ray data were collected in-house to a maximum resolution of 2 Å

  1. Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase from Escherichia coli

    International Nuclear Information System (INIS)

    Hor, Lilian; Dobson, Renwick C. J.; Dogovski, Con; Hutton, Craig A.; Perugini, Matthew A.

    2009-01-01

    Diaminopimelate (DAP) epimerase, an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary diffraction analysis of DAP epimerase from E. coli are reported. Diaminopimelate (DAP) epimerase (EC 5.1.1.7) catalyzes the penultimate step of lysine biosynthesis in bacteria and plants, converting l,l-diaminopimelate to meso-diaminopimelate. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DAP epimerase from Escherichia coli are presented. Crystals were obtained in space group P4 1 2 1 2 and diffracted to 2.0 Å resolution, with unit-cell parameters a = b = 89.4, c = 179.6 Å. Molecular replacement was conducted using Bacillus anthracis DAP epimerase as a search model and showed the presence of two molecules in the asymmetric unit, with an initial R free of 0.456 and R work of 0.416

  2. Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

    International Nuclear Information System (INIS)

    Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano; Macone, Alberto; Boffi, Alberto; Ilari, Andrea

    2008-01-01

    The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3 1 21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction

  3. Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli

    International Nuclear Information System (INIS)

    Gao, Benlian; Bertrand, Adam; Boles, William H.; Ellis, Holly R.; Mallett, T. Conn

    2005-01-01

    Crystallization of the native and SeMet FMN reductase protein of the E. coli alkanesulfonate monooxygenase two-component enzyme system is reported. The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 Å resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin

  4. Characterization, crystallization and preliminary X-ray analysis of the adhesive domain of SdrE from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Xiang, Hua; Gao, Fangfang; Wang, Dacheng; Liu, Jing; Hu, Jia; Zhang, Liqing; Li, Shentao; Deng, Xuming

    2010-01-01

    The adhesive domain of SdrE from Staphylococcus aureus was recombinantly expressed in Escherichia coli and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. The adhesive domain of SdrE from Staphylococcus aureus was recombinantly expressed in Escherichia coli. The purified protein was identified by SDS–PAGE and MALDI–TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 8000 as the primary precipitating agent. X-ray diffraction data were collected to 1.8 Å resolution from a single crystal of the protein. Preliminary X-ray analysis indicated that the crystal belonged to space group P1, with unit-cell parameters a = 40.714, b = 66.355, c = 80.827 Å, α = 111.19, β = 93.99, γ = 104.39°

  5. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

    OpenAIRE

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-01-01

    Nattokinase, a protein found in high levels in the traditional Japanese food natto, has been reported to have high thrombolytic activity. In the present study, the crystallization of native nattokinase and the collection of X-ray diffraction date from a nattokinase crystal to a resolution of 1.74 Å are reported.

  6. Purification, crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from Acinetobacter baumannii

    International Nuclear Information System (INIS)

    Park, Jeong Soon; Lee, Woo Cheol; Song, Jung Hyun; Kim, Seung Il; Lee, Je Chul; Cheong, Chaejoon; Kim, Hye-Yeon

    2012-01-01

    The crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from A. baumannii are reported. The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of l-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5′-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (ll-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Å and belonged to space group P3 1 or P3 2 , with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit

  7. Crystallization and preliminary X-ray analysis of argininosuccinate lyase from Streptococcus mutans

    International Nuclear Information System (INIS)

    Cao, Yan-Li; Li, Gui-Lan; Wang, Kai-Tuo; Zhang, Hong-Yin; Li, Lan-Fen

    2011-01-01

    Crystals of argininosuccinate lyase from S. mutans were obtained and X-ray data were collected to 2.5 Å resolution in space group R3. Argininosuccinate lyase (ASL) is an important enzyme in arginine synthesis and the urea cycle, which are highly conserved from bacteria to eukaryotes. The gene encoding Streptococcus mutans ASL (smASL) was amplified and cloned into expression vector pET28a. The recombinant smASL protein was expressed in a soluble form in Escherichia coli strain BL21 (DE3) and purified to homogeneity by two-step column chromatography. Crystals suitable for X-ray analysis were obtained and X-ray diffraction data were collected to a resolution of 2.5 Å. The crystals belonged to space group R3, with unit-cell parameters a = b = 254.5, c = 78.3 Å

  8. MraZ from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Melanie A.; Udell, Christian M.; Pal, Gour Pada; Jia, Zongchao, E-mail: jia@post.queensu.ca [Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6 (Canada)

    2005-04-01

    The crystallization and preliminary X-ray diffraction analysis of MraZ, formerly known as hypothetical protein YabB, from Escherichia coli K-12 is presented. The MraZ family of proteins, also referred to as the UPF0040 family, are highly conserved in bacteria and are thought to play a role in cell-wall biosynthesis and cell division. The murein region A (mra) gene cluster encodes MraZ proteins along with a number of other proteins involved in this complex process. To date, there has been no clear functional assignment provided for MraZ proteins and the structure of a homologue from Mycoplasma pneumoniae, MPN314, failed to suggest a molecular function. The b0081 gene from Escherichia coli that encodes the MraZ protein was cloned and the protein was overexpressed, purified and crystallized. This data is presented along with evidence that the E. coli homologue exists in a different oligomeric state to the MPN314 protein.

  9. MraZ from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

    International Nuclear Information System (INIS)

    Adams, Melanie A.; Udell, Christian M.; Pal, Gour Pada; Jia, Zongchao

    2005-01-01

    The crystallization and preliminary X-ray diffraction analysis of MraZ, formerly known as hypothetical protein YabB, from Escherichia coli K-12 is presented. The MraZ family of proteins, also referred to as the UPF0040 family, are highly conserved in bacteria and are thought to play a role in cell-wall biosynthesis and cell division. The murein region A (mra) gene cluster encodes MraZ proteins along with a number of other proteins involved in this complex process. To date, there has been no clear functional assignment provided for MraZ proteins and the structure of a homologue from Mycoplasma pneumoniae, MPN314, failed to suggest a molecular function. The b0081 gene from Escherichia coli that encodes the MraZ protein was cloned and the protein was overexpressed, purified and crystallized. This data is presented along with evidence that the E. coli homologue exists in a different oligomeric state to the MPN314 protein

  10. Expression, purification, crystallization and preliminary X-ray analysis of two arginine-biosynthetic enzymes from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Moradian, Fatemeh; Garen, Craig; Cherney, Leonid; Cherney, Maia; James, Michael N. G.

    2006-01-01

    Two enzymes responsible for arginine biosynthesis in M. tuberculosis were expressed in Escherichia coli, then purified to homogeneity. Preliminary X-ray analysis of diffraction-quality crystals grown from each enzyme are reported. The gene products of two open reading frames from Mycobacterium tuberculosis (Mtb) have been crystallized using the sitting-drop vapour-diffusion method. Rv1652 encodes a putative N-acetyl-γ-glutamyl-phosphate reductase (MtbAGPR), while the Rv1656 gene product is annotated as ornithine carbamoyltransferase (MtbOTC). Both MtbAGPR and MtbOTC were expressed in Escherichia coli, purified to homogeneity and crystallized. Native data for each crystal were collected to resolutions of 2.15 and 2.80 Å, respectively. Preliminary X-ray data are presented for both enzymes

  11. Purification, crystallization and preliminary X-ray diffraction studies of parakeet (Psittacula krameri) haemoglobin

    OpenAIRE

    Jaimohan, S. M.; Naresh, M. D.; Arumugam, V.; Mandal, A. B.

    2009-01-01

    Parakeet (Psittacula krameri) haemoglobin has been purified and crystallized under low salt buffered conditions. Preliminary analysis of the crystal that belonged to monoclinic system (C2) is reported.

  12. Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus)

    International Nuclear Information System (INIS)

    Sundaresan, S. S.; Ramesh, P.; Sivakumar, K.; Ponnuswamy, M. N.

    2009-01-01

    Purification, crystallization and preliminary X-ray analysis of haemoglobin from ostrich (Struthio camelus) has been carried out under 293 K temperature conditions. The ostrich is a large flightless bird which contains inositol tetrakisphosphate in erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure–function relationship of ostrich heamoglobin. Haemoglobin is a tetrameric protein that carries oxygen from the lungs to tissues and carbon dioxide from tissues back to the lungs. The oxygen-binding properties of haemoglobin are regulated through the binding of allosteric effectors. The respiratory system of avian species is unique and complex in nature when compared with that of mammals. In avian species, inositol pentaphosphate (inositol-P 5 ) is present in the erythrocytes of the adult and is thought to be the major factor responsible for the relatively high oxygen affinity of the whole blood. The ostrich (Struthio camelus) is a large flightless bird which contains inositol tetrakisphosphate (inositol-P 4 ) in its erythrocytes and its whole blood oxygen affinity is higher. Efforts have been made to explore the structure–function relationship of ostrich haemoglobin. Ostrich haemoglobin was purified using ion-exchange chromatography. Haemoglobin crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant in 50 mM phosphate buffer pH 7.2. Data were collected using a MAR345 image-plate detector system. The crystals of ostrich haemoglobin diffracted to 2.2 Å resolution. They belonged to the orthorhombic space group P2 1 2 1 2 1 with one whole biological molecule in the asymmetric unit; the unit-cell parameters were a = 80.93, b = 81.68, c = 102.05 Å

  13. Preliminary morphological and X-ray diffraction studies of the crystals of the DNA cetyltrimethylammonium salt.

    Science.gov (United States)

    Osica, V D; Pyatigorskaya, T L; Polyvtsev, O F; Dembo, A T; Kliya, M O; Vasilchenko, V N; Verkin, B I; Sukharevskya, B Y

    1977-04-01

    Double-stranded DNA molecules (molecular weight 2.5 X 10(5) - 5 X 10(5) daltons) have been crystallized from water-salt solutions as cetyltrimethylammonium salts (CTA-DNA). Variation of crystallization conditions results in a production of different types of CTA-DNA crystals: spherulits, dendrites, needle-shaped and faceted rhombic crystals, the latter beeing up to 0.3 mm on a side. X-ray diffraction data indicate that DNA molecules in the crystals form a hexagonal lattice which parameters vary slightly with the morphological type of the crystal. Comparison of the melting curves of the DNA preparation before and after crystallization suggests that DNA molecules are partially fractionated in the course of crystallization. Crystals of the CTA-DNA-proflavine complex have also been obtained.

  14. Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase

    International Nuclear Information System (INIS)

    Gunn, Natalie J.; Gorman, Michael A.; Dobson, Renwick C. J.; Parker, Michael W.; Mulhern, Terrence D.

    2011-01-01

    The Src-homology 2 (SH2) domain of Csk-family protein tyrosine kinases acts as a conformational switch to regulate their catalytic activity, which in turn promotes the inhibition of their proto-oncogenic targets, the Src-family kinases. Here, the expression, purification, small-angle X-ray scattering and preliminary diffraction analysis of the SH2 domain of the Csk-homologous kinase is reported. The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a = 25.8, b = 34.6, c = 63.2 Å, β = 99.4°

  15. Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum

    International Nuclear Information System (INIS)

    Ashby, Jamie A.; Stevenson, Clare E. M.; Maule, Andrew J.; Lawson, David M.

    2009-01-01

    Crystals of N-terminally truncated eIF4E from pea were obtained and X-ray data were recorded in-house to a resolution of 2.2 Å. Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (ΔN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 Å from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection

  16. Crystallization and preliminary X-ray diffraction analysis of alanine racemase from Pseudomonas putida YZ-26

    International Nuclear Information System (INIS)

    Liu, Junlin; Feng, Lei; Shi, Yawei; Feng, Wei

    2012-01-01

    A recombinant alanine racemase from the Pseudomonas putida YZ-26, has been crystallized by the sitting-drop vapor-diffusion method and X-ray diffraction data were collected to 2.4 Å. A recombinant form of alanine racemase (Alr) from Pseudomonas putida YZ-26 has been crystallized by the sitting-drop vapour diffusion method. X-ray diffraction data were collected to 2.4 Å resolution. The crystals belong to the space group C222 1 , with unit-cell parameters a = 118.08, b = 141.86, c = 113.83 Å, and contain an Alr dimer in the asymmetric unit. The Matthews coefficient and the solvent content were calculated to be 2.8 Å 3 Da −1 and approximately 50%, respectively

  17. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

    Science.gov (United States)

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-12-01

    Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, β=95.2°. Diffraction images were processed to a resolution of 1.74 Å with an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

  18. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

    International Nuclear Information System (INIS)

    Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

    2010-01-01

    Nattokinase, a protein found in high levels in the traditional Japanese food natto, has been reported to have high thrombolytic activity. In the present study, the crystallization of native nattokinase and the collection of X-ray diffraction date from a nattokinase crystal to a resolution of 1.74 Å are reported. Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase

  19. Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

    Energy Technology Data Exchange (ETDEWEB)

    Yanagisawa, Yasuhide [Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025 (Japan); Chatake, Toshiyuki [Research Reactor Institute, Kyoto University, Asashironishi 2, Kumatori, Sennan, Osaka 590-0494 (Japan); Chiba-Kamoshida, Kaori [National Institute of Advanced Industrial Science and Technology (AIST), Umezono 1-1-1, Tsukuba, Ibaraki 305-8568 (Japan); Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki [Kurashiki University of Science and Arts, Nishinoura 2640, Tsurajima-cho, Kurashiki, Okayama 712-8505 (Japan); Yasuda, Ichiro [Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025 (Japan); Morimoto, Yukio [Research Reactor Institute, Kyoto University, Asashironishi 2, Kumatori, Sennan, Osaka 590-0494 (Japan); Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025 (Japan)

    2010-12-01

    Nattokinase, a protein found in high levels in the traditional Japanese food natto, has been reported to have high thrombolytic activity. In the present study, the crystallization of native nattokinase and the collection of X-ray diffraction date from a nattokinase crystal to a resolution of 1.74 Å are reported. Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R{sub merge} of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

  20. Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides

    International Nuclear Information System (INIS)

    Navarro, Marcos Vicente de A. S.; Vierira, Débora F.; Nagem, Ronaldo A. P.; Araújo, Ana Paula U. de; Oliva, Maria Luiza V.; Garratt, Richard C.

    2005-01-01

    Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges. A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V M = 2.5 Å 3 Da −1 ). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method

  1. Crystallization and preliminary X-ray crystallographic analysis of dioscorin from Dioscorea japonica

    International Nuclear Information System (INIS)

    Xue, You-Lin; Miyakawa, Takuya; Sawano, Yoriko; Tanokura, Masaru

    2012-01-01

    Dioscorin from D. japonica was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The dioscorin crystal diffracted X-rays to 2.11 Å resolution. Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and monodehydroascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulation via the toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytes ex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1–246) with an additional His 6 tag at the N-terminus was expressed in Escherichia coli Rosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-ray source. The crystal belonged to space group C222 1 , with unit-cell parameters a = 83.5, b = 156.8, c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit

  2. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    International Nuclear Information System (INIS)

    Nascimento, Alessandro S.; Ferrarezi, Thiago; Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A.; Polikarpov, Igor

    2006-01-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP + reductase. Ferredoxin-NADP + reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source

  3. Crystallization and preliminary X-ray diffraction studies of ferredoxin reductase from Leptospira interrogans

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Alessandro S.; Ferrarezi, Thiago [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil); Catalano-Dupuy, Daniela L.; Ceccarelli, Eduardo A. [Facultad de Ciencias Bioquímicas y Farmacéuticas, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario (Argentina); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Saocarlense 400, São Carlos, SP, 13560-970 (Brazil)

    2006-07-01

    Crystals adequate for X-ray diffraction analysis have been prepared from L. interrogans ferredoxin-NADP{sup +} reductase. Ferredoxin-NADP{sup +} reductase (FNR) is an FAD-containing enzyme that catalyzes electron transfer between NADP(H) and ferredoxin. Here, results are reported of the recombinant expression, purification and crystallization of FNR from Leptospira interrogans, a parasitic bacterium of animals and humans. The L. interrogans FNR crystals belong to a primitive monoclinic space group and diffract to 2.4 Å resolution at a synchrotron source.

  4. Purification, crystallization and preliminary X-ray characterization of a haemagglutinin from the seeds of Jatropha curcas

    International Nuclear Information System (INIS)

    Nair, Divya N.; Suresh, C. G.; Singh, Desh Deepak

    2011-01-01

    A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P2 1 2 1 2 1 and the crystals diffracted to 2.8 Å resolution at 103 K. The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P2 1 2 1 2 1 . The crystals diffracted to 2.8 Å resolution at 103 K

  5. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    Energy Technology Data Exchange (ETDEWEB)

    Nithianantham, Stanley [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States); Xu, Minghua [Department of Biological Sciences, School of Dentistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4905 (United States); Wu, Nan [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States); Han, Yiping W., E-mail: ywh2@case.edu [Department of Biological Sciences, School of Dentistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4905 (United States); Department of Pathology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106 (United States); Shoham, Menachem, E-mail: ywh2@case.edu [Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4935 (United States)

    2006-12-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6{sub 1} or P6{sub 5}, and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum.

  6. Crystallization and preliminary X-ray data of the FadA adhesin from Fusobacterium nucleatum

    International Nuclear Information System (INIS)

    Nithianantham, Stanley; Xu, Minghua; Wu, Nan; Han, Yiping W.; Shoham, Menachem

    2006-01-01

    The FadA adhesin from F. nucleatum, which is involved in bacterial attachment and invasion of human oral epithelial cells, has been crystallized in space group P6 1 or P6 5 , and X-ray data have been collected to 1.9 Å resolution. Fusobacterium nucleatum is a Gram-negative anaerobe prevalent in the oral cavity that is associated with periodontal disease, preterm birth and infections in other parts of the human body. The bacteria attach to and invade epithelial and endothelial cells in the gum tissue and elsewhere via a 13.7 kDa adhesin protein FadA (Fusobacterium adhesin A). FadA exists in two forms: the intact form (pre-FadA), consisting of 129 amino acids, and the mature form (mFadA), which lacks an 18-residue signal sequence. Both forms have been expressed in Escherichia coli and purified. mFadA has been crystallized. The crystals belong to the hexagonal space group P6 1 or P6 5 , with unit-cell parameters a = b = 59.3, c = 125.7 Å and one molecule per asymmetric unit. The crystals exhibit an unusually high solvent content of 74%. Synchrotron X-ray data have been collected to 1.9 Å. The crystals are suitable for X-ray structure determination. The crystal structure of FadA may provide a basis for the development of therapeutic agents to combat periodontal disease and other infections associated with F. nucleatum

  7. Production, crystallization and preliminary X-ray diffraction analysis of the allergen Can f 2 from Canis familiaris

    International Nuclear Information System (INIS)

    Madhurantakam, Chaithanya; Nilsson, Ola B.; Jönsson, Klas; Grönlund, Hans; Achour, Adnane

    2009-01-01

    The recombinant form of the allergen Can f 2 from C. familiaris was produced, isolated and crystallized in two different forms. Preliminary X-ray diffraction analyses are reported for the two crystal forms of Can f 2. The allergen Can f 2 from dog (Canis familiaris) present in saliva, dander and fur is an important cause of allergic sensitization worldwide. Here, the production, isolation, crystallization and preliminary X-ray diffraction analysis of two crystal forms of recombinant Can f 2 are reported. The first crystal form belonged to space group C222, with unit-cell parameters a = 68.7, b = 77.3, c = 65.1 Å, and diffracted to 1.55 Å resolution, while the second crystal form belonged to space group C2, with unit-cell parameters a = 75.7, b = 48.3, c = 68.7 Å, β = 126.5°, and diffracted to 2.1 Å resolution. Preliminary data analysis indicated the presence of a single molecule in the asymmetric unit for both crystal forms

  8. Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Kim, Hyung-Wook; Han, Byung Woo; Yoon, Hye-Jin; Yang, Jin Kuk; Lee, Byung Il; Lee, Hyung Ho; Ahn, Hyung Jun; Suh, Se Won

    2002-10-01

    Peptide deformylase (PDF) from the pathogenic bacterium Pseudomonas aeruginosa has been overexpressed in Escherichia coli and crystallized in the presence of its inhibitor actinonin at 297 K using polyethylene glycol (PEG) 4000 as a precipitant. The diffraction limit and the spot shape of the crystals could be slightly improved by the crystal annealing/dehydration procedure. X-ray diffraction data to 1.85 A have been collected using synchrotron radiation. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.75, b = 74.46, c = 77.18 A. The asymmetric unit contains two subunits of peptide deformylase, with a corresponding crystal volume per protein mass (V(M)) of 2.45 A(3) Da(-1) and a solvent content of 49.8%.

  9. Crystallization and preliminary X-ray diffraction analysis of HML, a lectin from the red marine alga Hypnea musciformis

    International Nuclear Information System (INIS)

    Nagano, Celso S.; Gallego del Sol, Francisca; Cavada, Benildo S.; Nascimento, Kyria Santiago Do; Nunes, Eudismar Vale; Sampaio, Alexandre H.; Calvete, Juan J.

    2005-01-01

    The crystallization and preliminary X-ray diffraction analysis of a red marine alga lectin isolated from H. musciformis is reported. HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P2 1 2 1 2 1 grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 Å resolution. HML is the first marine alga lectin to be crystallized

  10. Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii

    International Nuclear Information System (INIS)

    Arnfors, Linda; Hansen, Thomas; Meining, Winfried; Schönheit, Peter; Ladenstein, Rudolf

    2005-01-01

    Nucleoside kinase from the hyperthermophilic archaeon M. jannaschii is a member of the PFK-B family which belongs to the ribokinase superfamily. Here, its expression, purification, crystallization and preliminary X-ray analysis are described. Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg 2+ . The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 Å for the apoenzyme crystals and 1.93 Å for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer

  11. Cloning, expression, purification, crystallization and preliminary X-ray analysis of the pilus-associated sortase C from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Neiers, F.; Madhurantakam, C.; Fälker, S.; Normark, S.; Henriques-Normark, B.; Achour, A.

    2008-01-01

    Crystallization conditions and preliminary X-ray diffraction analysis of the S. pneumoniae-derived pilus-associated protein sortase C are reported. The pilus-associated sortase C from Streptococcus pneumoniae (SrtC or Srt-2) acts as a polymerase for the pilus subunit proteins RrgA and RrgB. Here, the crystallization and preliminary X-ray diffraction analysis of three crystal forms of SrtC are reported. One crystal form belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.9, b = 96.9, c = 98.9 Å, α = β = γ = 90°. The other two crystal forms belong to space group P222, with unit-cell parameters a = 48.8, b = 97.2, c = 99.2 Å, α = β = γ = 90° and a = 48.6, b = 96.5, c = 98.8 Å, α = β = γ = 90°, respectively. Preliminary analysis indicates the presence of two molecules in the asymmetric unit of the crystal for all three forms

  12. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

    Energy Technology Data Exchange (ETDEWEB)

    Balasubramanian, Anuradha; Ponnuraj, Karthe, E-mail: pkarthe@hotmail.com [Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025 (India)

    2008-07-01

    Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å.

  13. Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsG

    International Nuclear Information System (INIS)

    Jens, Jason; Raghunathan, Kannan; Vago, Frank; Arvidson, Dennis

    2009-01-01

    Recombinant V. cholerae EpsG has been expressed, purified and crystallized. The crystals diffracted to 2.26 Å resolution. EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 Å and belonged to space group P2 1 , with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 Å

  14. Purification, crystallization and preliminary X-ray analysis of urease from jack bean (Canavalia ensiformis)

    International Nuclear Information System (INIS)

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2009-01-01

    Jack bean urease was purified and crystallized and X-ray diffraction data were collected to 2.05 Å resolution. Plant urease is a seed protein that is common in most legumes. It is also common in many bacteria and fungi and several species of yeast. Urease allows organisms to use exogenous and internally generated urea as a nitrogen source by catalyzing the hydrolysis of urea to ammonia and carbon dioxide. Urease from jack bean meal was purified to electrophoretic homogeneity using a series of steps involving acetone precipitation and size-exclusion and ion-exchange chromatography. The jack bean urease was crystallized and the resulting crystals diffracted to 2.05 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6 3 22, with unit-cell parameters a = b = 138.57, c = 198.36 Å

  15. Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

    International Nuclear Information System (INIS)

    Balasubramanian, Anuradha; Ponnuraj, Karthe

    2008-01-01

    Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized and the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å

  16. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    Energy Technology Data Exchange (ETDEWEB)

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  17. Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus

    International Nuclear Information System (INIS)

    Martin, Stanton L.; Guenther, Richard H.; Sit, Tim L.; Swartz, Paul D.; Meilleur, Flora; Lommel, Steven A.; Rose, Robert B.

    2010-01-01

    Virions of red clover necrotic mosaic virus have been purified and crystallized. The space group was determined to be I23, with unit-cell parameter a = 377.8 Å. The crystals diffracted to 4 Å resolution. Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Å resolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Å in the analysis presented here

  18. Crystallization and preliminary X-ray analysis of cecropin B from Bombyx mori

    International Nuclear Information System (INIS)

    Liu, Zhongyuan; Zhou, Qiangjun; Mao, Xinfang; Zheng, Xiangdong; Guo, Jiubiao; Zhang, Fuchun; Wen, Tingyi; Pang, Hai

    2010-01-01

    Cecropin B derived from the hemolymph of Bombyx mori has been crystallized by the hanging-drop vapour-diffusion method. The crystal diffracted to 1.43 Å resolution using X-ray radiation. Cecropin B is a 37-residue cationic antimicrobial peptide derived from the haemolymph of Bombyx mori. The precise mechanism by which cecropins exert their antimicrobial and cytolytic activities is not well understood. Crystals of cecropin B were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant at 289 K. The crystal diffracted to 1.43 Å resolution using X-ray radiation and belonged to the orthorhombic space group P1, with unit-cell parameters a = 15.08, b = 22.75, c = 30.20 Å, α = 96.9, β = 103.1, γ = 96.5°. The asymmetric unit contained only one molecule of cecropin B, with a calculated Matthews coefficient of 2.48 Å 3 Da −1 and a solvent content of 50.4%

  19. Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruzi

    International Nuclear Information System (INIS)

    Matoba, Kazuaki; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Inaoka, Daniel Ken; Kita, Kiyoshi; Harada, Shigeharu

    2009-01-01

    Aspartate transcarbamoylase, the second enzyme of the de novo pyrimidine-biosynthetic pathway, from T. cruzi has been purified and crystallized for X-ray structure analysis. Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and l-aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a complex with carbamoyl phosphate at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. Ligand-free crystals (space group P1, unit-cell parameters a = 78.42, b = 79.28, c = 92.02 Å, α = 69.56, β = 82.90, γ = 63.25°) diffracted X-rays to 2.8 Å resolution, while those cocrystallized with carbamoyl phosphate (space group P2 1 , unit-cell parameters a = 88.41, b = 158.38, c = 89.00 Å, β = 119.66°) diffracted to 1.6 Å resolution. The presence of two homotrimers in the asymmetric unit (38 kDa × 6) gives V M values of 2.3 and 2.5 Å 3 Da −1 for the P1 and P2 1 crystal forms, respectively

  20. Crystallization and preliminary X-ray diffraction analysis of recombinant hepatitis E virus-like particle

    International Nuclear Information System (INIS)

    Wang, Che-Yen; Miyazaki, Naoyuki; Yamashita, Tetsuo; Higashiura, Akifumi; Nakagawa, Atsushi; Li, Tian-Cheng; Takeda, Naokazu; Xing, Li; Hjalmarsson, Erik; Friberg, Claes; Liou, Der-Ming; Sung, Yen-Jen; Tsukihara, Tomitake; Matsuura, Yoshiharu; Miyamura, Tatsuo; Cheng, R. Holland

    2008-01-01

    A recombinant virus-like particle that is a potential oral hepatitis E vaccine was crystallized. Diffraction data were collected to 8.3 Å resolution and the X-ray structure was phased with the aid of a low-resolution density map determined using cryo-electron microscopy data. Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3 protein (residue 70–123) fused to the N-terminus of the ORF2 protein (residues 112–608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 Å resolution. The crystals belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 337, b = 343, c = 346 Å, α = β = γ = 90°, and contain one particle per asymmetric unit

  1. Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin

    International Nuclear Information System (INIS)

    Alfaro, Elisa; Valle Sosa, Liliana del; Sanoguet, Zuleika; Pastrana-Ríos, Belinda; Schreiter, Eric R.

    2008-01-01

    C. reinhardtii centrin, an EF-hand calcium-binding protein localized to the microtubule-organizing center of eukaryotic organisms, has been crystallized in the presence of the model peptide melittin. X-ray diffraction data were collected to 2.2 Å resolution. Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 Å resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 Å, and are likely to belong to space group P2 1 2 1 2

  2. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinase

    International Nuclear Information System (INIS)

    Balogun, Emmanuel Oluwadare; Inaoka, Daniel Ken; Kido, Yasutoshi; Shiba, Tomoo; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, Masayuki; Matsuoka, Shigeru; Michels, Paul A. M.; Harada, Shigeharu; Kita, Kiyoshi

    2010-01-01

    Glycerol kinase from human African trypanosomes has been purified and crystallized for X-ray structure analysis. In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His 6 tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75 Å resolution indicated that the crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 63.84, b = 121.50, c = 154.59 Å. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (V M ) of 2.5 Å 3 Da −1 , corresponding to 50% solvent content

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum

    International Nuclear Information System (INIS)

    Trincão, José; Sousa Silva, Marta; Barata, Lídia; Bonifácio, Cecília; Carvalho, Sandra; Tomás, Ana Maria; Ferreira, António E. N.; Cordeiro, Carlos; Ponces Freire, Ana; Romão, Maria João

    2006-01-01

    A glyoxalase II from L. infantum was cloned, purified and crystallized and its structure was solved by X-ray crystallography. In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222 1 (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyond 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypano-somatids

  4. Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Lu, Lu; Nan, Jie; Mi, Wei; Wei, Chun-Hong; Li, Lan-Fen; Li, Yi

    2010-01-01

    Tubulin-folding cofactor A from A. thaliana has been crystallized and preliminarily analyzed using X-ray diffraction. Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the β-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289 K. The crystal diffracted to 1.6 Å resolution using synchrotron radiation and belonged to space group I4 1 , with unit-cell parameters a = 55.0, b = 55.0, c = 67.4 Å

  5. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulator AcrR from Escherichia coli

    International Nuclear Information System (INIS)

    Li, Ming; Qiu, Xi; Su, Chih-Chia; Long, Feng; Gu, Ruoyu; McDermott, Gerry; Yu, Edward W.

    2006-01-01

    The transcriptional regulator AcrR from Escherichia coli has been cloned, overexpressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.5 Å. This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5 Å. The space group was determined to be P3 2 , with unit-cell parameters a = b = 46.61, c = 166.16 Å

  6. Expression, crystallization and preliminary X-ray crystallographic analysis of human agmatinase

    International Nuclear Information System (INIS)

    Kim, Kyoung Hoon; Ahn, Hyung Jun; Kim, Do Jin; Lee, Hyung Ho; Ha, Jun-Yong; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2005-01-01

    Human agmatinase (Ala36–Val352) was overexpressed and crystallized, and X-ray diffraction data were collected to 2.49 Å. Agmatine, which results from the decarboxylation of l-arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putresine and higher polyamines (spermidine and spermine). Recent studies indicate that agmatine can have several important biochemical effects in humans, ranging from effects on the central nervous system to cell proliferation in cancer and viral replication. Agmatinase catalyses the hydrolysis of agmatine to putresine and urea and is a major target for drug action and development. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Human agmatinase (residues Ala36–Val352) has been overexpressed as a fusion with both N- and C-terminal purification tags in Escherichia coli and crystallized in the presence of Mn 2+ and 1,6-diaminohexane at 297 K using polyethylene glycol 4000 as a precipitant. X-ray diffraction data were collected at 100 K to 2.49 Å from a flash-frozen crystal. The crystals are tetragonal, belonging to space group P4 2 , with unit-cell parameters a = b = 114.54, c = 125.65 Å, α = β = γ = 90°. Three monomers are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (V M ) of 3.66 Å 3 Da −1 and a solvent content of 66.4%

  7. Expression, crystallization and preliminary X-ray crystallographic analysis of human agmatinase

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung Hoon; Ahn, Hyung Jun; Kim, Do Jin; Lee, Hyung Ho; Ha, Jun-Yong; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2005-10-01

    Human agmatinase (Ala36–Val352) was overexpressed and crystallized, and X-ray diffraction data were collected to 2.49 Å. Agmatine, which results from the decarboxylation of l-arginine by arginine decarboxylase, is a metabolic intermediate in the biosynthesis of putresine and higher polyamines (spermidine and spermine). Recent studies indicate that agmatine can have several important biochemical effects in humans, ranging from effects on the central nervous system to cell proliferation in cancer and viral replication. Agmatinase catalyses the hydrolysis of agmatine to putresine and urea and is a major target for drug action and development. The human agmatinase gene encodes a 352-residue protein with a putative mitochondrial targeting sequence at the N-terminus. Human agmatinase (residues Ala36–Val352) has been overexpressed as a fusion with both N- and C-terminal purification tags in Escherichia coli and crystallized in the presence of Mn{sup 2+} and 1,6-diaminohexane at 297 K using polyethylene glycol 4000 as a precipitant. X-ray diffraction data were collected at 100 K to 2.49 Å from a flash-frozen crystal. The crystals are tetragonal, belonging to space group P4{sub 2}, with unit-cell parameters a = b = 114.54, c = 125.65 Å, α = β = γ = 90°. Three monomers are likely to be present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.66 Å{sup 3} Da{sup −1} and a solvent content of 66.4%.

  8. Crystallization and preliminary X-ray crystallographic analysis of dioscorin from Dioscorea japonica.

    Science.gov (United States)

    Xue, You-Lin; Miyakawa, Takuya; Sawano, Yoriko; Tanokura, Masaru

    2012-02-01

    Dioscorin, the major tuber storage protein in yam, has been reported to possess carbonic anhydrase, trypsin inhibitor, angiotensin-converting enzyme (ACE) inhibitor, free-radical scavenger, dehydroascorbate reductase and monodehydroascorbate reductase activities. Recent research has also found that dioscorin can enhance immune modulation via the toll-like receptor 4 (TLR-4) signal transduction pathway in RAW 264.7 cells, murine bone-marrow cells and human monocytes ex vivo. Resolving the structure of dioscorin would help in better understanding its activities and would provide clues to understanding the mechanism of its multiple functions. The full-length protein (residues 1-246) with an additional His(6) tag at the N-terminus was expressed in Escherichia coli Rosetta (DE3) cells. After His-tag cleavage and purification, the protein was crystallized by the sitting-drop vapour-diffusion method at 278 K. An X-ray diffraction data set was collected to a resolution of 2.11 Å using a synchrotron X-ray source. The crystal belonged to space group C222(1), with unit-cell parameters a = 83.5, b = 156.8, c = 83.6 Å, and was estimated to contain two protein molecules per asymmetric unit.

  9. Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, Marcos Vicente de A. S.; Vierira, Débora F. [Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, CEP 13560-970, São Carlos, SP (Brazil); Nagem, Ronaldo A. P. [Biochemistry and Immunology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Antônio Carlos 6627, Caixa Postal 486, CEP 31270-901, Belo Horizonte, MG (Brazil); Araújo, Ana Paula U. de [Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, CEP 13560-970, São Carlos, SP (Brazil); Oliva, Maria Luiza V. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio 100, CEP 04044-020, São Paulo, SP (Brazil); Garratt, Richard C., E-mail: richard@if.sc.usp.br [Institute of Physics of São Carlos, University of São Paulo, Avenida Trabalhador Sãocarlense 400, CEP 13560-970, São Carlos, SP (Brazil)

    2005-10-01

    Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges. A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V{sub M} = 2.5 Å{sup 3} Da{sup −1}). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.

  10. Crystallization and preliminary X ray analysis of nucleoside diphosphate kinase 1 from T. cruzi

    International Nuclear Information System (INIS)

    Gomez Barroso, J.A.; Aguilar, C.F.; Miranda, M.R.; Pereira, C.A.

    2009-01-01

    Introduction: Trypanosoma cruzi is the etiologic agent of Chagas disease. The Nucleoside diphosphate kinases (NDPKs) are enzymes involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform. The objective of this work is obtaining protein's crystals, diffract and process the data for tridimensional structure resolution. Materials and Methods: TcNDPK1 was expressed in E. coli as a fusion protein with Nterminal His-tag. TcNDPK1 was overexpressed and purified by FPLC. Crystallization was assayed by sitting drop and hanging drop vapor diffusion method. Crystals was frozen and diffracted on synchrotron x-ray radiation in Campinas (Brasil). The data set collected was reduced and merged using MOSFLM and SCALA programs. Results and Discussion: His-TcNDPK was overexpressed, purified and crystallized. The crystals are diffracted and collected the data to 3.5A. The crystals belong to the trigonal space group P3, with unit cell parameters a=127.94, b=127.84, c=275.49. Structure determination is under way. These results will provide relevant information that could be the first step in rational drug design for treating Chagas disease.(authors)

  11. Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Schwanniomyces occidentalis

    International Nuclear Information System (INIS)

    Polo, Aitana; Álvaro-Benito, Miguel; Fernández-Lobato, María; Sanz-Aparicio, Julia

    2009-01-01

    The invertase from Schwanniomyces occidentalis has been expressed in Saccharomyces cerevisiae, purified and crystallized. The wild-type enzyme was also purified and crystallized and diffraction data were collected to 2.9 Å resolution. Schwanniomyces occidentalis invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The enzyme has been expressed in Saccharomyces cerevisiae. Recombinant and wild-type forms, which showed different glycosylation patterns, were crystallized by vapour-diffusion methods. Although crystallization trials were conducted on both forms of the protein, crystals suitable for X-ray crystallographic analyses were only obtained from the wild-type enzyme. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 105.78, b = 119.49, c = 137.68 Å. A diffraction data set was collected using a synchrotron source. Self-rotation function and sedimentation-velocity experiments suggested that the enzyme was dimeric with twofold symmetry

  12. Purification, crystallization and preliminary X-ray diffraction analysis of human Gadd45γ

    International Nuclear Information System (INIS)

    Zhang, Wenzheng; Zhao, Mingzhuo; Li, Jianhui; Li, Xuemei; Zeng, ZongHao; Rao, Zihe

    2008-01-01

    The human Gadd45γ protein has been crystallized as a prelude towards determination of its three-dimensional structure by X-ray crystallography. Gadd45, MyD118 and CR6 (also termed Gadd45α, Gadd45β and Gadd45γ, respectively) comprise a family of proteins that play important roles in negative growth control, maintenance of genomic stability, DNA repair, cell-cycle control and apoptosis. Recombinant human Gadd45γ and its selenomethionine derivative were expressed in an Escherichia coli expression system and purified; they were then crystallized using the hanging-drop vapour-diffusion method. Diffraction-quality crystals were grown at 291 K using PEG 3350 as precipitant. Using synchrotron radiation, the best diffraction data were collected to 2.3 Å resolution for native crystals at 100 K; selenomethionyl derivative data were collected to 3.3 Å resolution. All the crystals belonged to space group I2 1 3, with approximate unit-cell parameters a = b = c = 126 Å

  13. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-01-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  14. Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinase from Streptococcus mutans

    International Nuclear Information System (INIS)

    Li, Hui; Zhao, Hongyan; Zhu, Laikuan; Hong, Lihua; Zhang, Hong; Lin, Fanjing; Xu, Chunyan; Li, Shentao; Zhang, Zhimin

    2012-01-01

    S-Ribosylhomocysteinase (LuxS) encoded by the LuxS gene from Streptococcus mutans was solubly expressed in Escherichia coli, purified and crystallized. Diffraction by the crystal extended to 2.4 Å resolution. S-Ribosylhomocysteinase (LuxS) encoded by the luxS gene from Streptococcus mutans plays a crucial role in the quorum-sensing system. LuxS was solubly expressed in Escherichia coli with high yield. The purity of the purified target protein, which was identified by SDS–PAGE and MALDI–TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space group C222 1 , with unit-cell parameters a = 55.3, b = 148.7, c = 82.8 Å

  15. Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

    International Nuclear Information System (INIS)

    Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko; Sakai, Hiroshi

    2005-01-01

    This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6 2 22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6 2 22, with unit-cell parameters a = b = 145.97, c = 118.03 Å

  16. Overexpression, purification, crystallization and preliminary X-ray crystal analysis of Bacillus pallidusd-arabinose isomerase

    International Nuclear Information System (INIS)

    Takeda, Kosei; Yoshida, Hiromi; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2008-01-01

    Recombinant B. pallidusd-arabinose isomerase was crystallized and diffraction data were collected to 2.3 Å resolution. d-Arabinose isomerase catalyzes the isomerization of d-arabinose to d-ribulose. Bacillus pallidusd-arabinose isomerase has broad substrate specificity and can catalyze the isomerization of d-arabinose, l-fucose, l-xylose, l-galactose and d-altrose. Recombinant B. pallidusd-arabinose isomerase was overexpressed, purified and crystallized. A crystal of the enzyme was obtained by the sitting-drop method at room temperature and belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 144.9, b = 127.9, c = 109.5 Å. Diffraction data were collected to 2.3 Å resolution

  17. Crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5

    International Nuclear Information System (INIS)

    Frank, Filipp; Virgili, Geneviève; Sonenberg, Nahum; Nagar, Bhushan

    2009-01-01

    The MIF4G domain of DAP5 was crystallized in two distinct crystal forms. Diffraction patterns have been analyzed and preliminary analysis, including molecular replacement, is presented here. Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented

  18. Crystallization and preliminary X-ray analysis of human S100A13

    Energy Technology Data Exchange (ETDEWEB)

    Imai, Fabiana Lica [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Nagata, Koji [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Yonezawa, Naoto [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Yu, Jinyan; Ito, Eriko; Kanai, Saeko [Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan); Tanokura, Masaru [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Nakano, Minoru, E-mail: mnakano@faculty.chiba-u.jp [Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522 (Japan)

    2006-11-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2{sub 1}2{sub 1}2{sub 1}.

  19. Crystallization and preliminary X-ray analysis of human S100A13

    International Nuclear Information System (INIS)

    Imai, Fabiana Lica; Nagata, Koji; Yonezawa, Naoto; Yu, Jinyan; Ito, Eriko; Kanai, Saeko; Tanokura, Masaru; Nakano, Minoru

    2006-01-01

    Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P2 1 2 1 2 1 and diffracted to a resolution of 1.8 Å. S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P2 1 2 1 2 1

  20. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    Gómez Barroso, J. A.; Pereira, H.; Miranda, M.; Pereira, C.; Garratt, R. C.; Aguilar, C. F.

    2010-01-01

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl 2 , 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  1. Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2

    International Nuclear Information System (INIS)

    Ascher, David B.; Polekhina, Galina; Parker, Michael W.

    2012-01-01

    The luminal domain of human endoplasmic reticulum aminopeptidase 2 has been expressed, purified and crystallized. The crystals belonged to the orthorhombic space group P2 1 2 1 2 and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house source. Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures

  2. Crystallization and preliminary X-ray diffraction analysis of a tRNASer acceptor-stem microhelix

    International Nuclear Information System (INIS)

    Förster, Charlotte; Krauss, Norbert; Brauer, Arnd B. E.; Szkaradkiewicz, Karol; Brode, Svenja; Hennig, Klaus; Fürste, Jens P.; Perbandt, Markus; Betzel, Christian; Erdmann, Volker A.

    2006-01-01

    In order to investigate tRNA identity elements, an elongator tRNA Ser acceptor-stem helix was crystallized and a data set was collected to 1.8 Å resolution aiming at a comparison with the corresponding region in suppressor tRNA Sec . In order to understand elongator tRNA Ser and suppressor tRNA Sec identity elements, the respective acceptor-stem helices have been synthesized and crystallized in order to analyse and compare their structures in detail at high resolution. The synthesis, crystallization and preliminary X-ray diffraction results for a seven-base-pair tRNA Ser acceptor-stem helix are presented here. Diffraction data were collected to 1.8 Å, applying synchrotron radiation and cryogenic cooling. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 36.14, b = 38.96, c = 30.81 Å, β = 110.69°

  3. Purification, crystallization and preliminary X-ray crystallographic analysis of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa

    International Nuclear Information System (INIS)

    Lin, Yi-Hung; Peng, Wen-Yan; Huang, Yen-Chieh; Guan, Hong-Hsiang; Hsieh, Ying-Cheng; Liu, Ming-Yih; Chang, Tschining; Chen, Chun-Jung

    2006-01-01

    The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported. Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P2 1 2 1 2, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%

  4. Crystallization and preliminary X-ray diffraction analysis of motif N from Saccharomyces cerevisiae Dbf4

    International Nuclear Information System (INIS)

    Matthews, Lindsay A.; Duong, Andrew; Prasad, Ajai A.; Duncker, Bernard P.; Guarné, Alba

    2009-01-01

    To understand the role of the Cdc7–Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. The Cdc7–Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7–Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7–Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 Å resolution and structure determination is currently under way

  5. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Radzimanowski, Jens; Ravaud, Stephanie; Schott, Andrea; Strahl, Sabine; Sinning, Irmgard

    2009-01-01

    Overexpression, purification, crystallization and preliminary X-ray diffraction of the stromal-cell-derived factor 2-like protein of Arabidopsis thaliana are reported. The crystals belonged to the space group P6 1 and diffracted to 1.95 Å resolution. The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 Å resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6 1 , with unit-cell parameters a = b = 96.1, c = 69.3 Å

  6. Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

    International Nuclear Information System (INIS)

    Garcia, Wanius; Travensolo, Regiane F.; Rodrigues, Nathalia C.; Muniz, João R. C.; Caruso, Célia S.; Lemos, Eliana G. M.; Araujo, Ana Paula U.; Carrilho, Emanuel

    2008-01-01

    Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source

  7. Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, Wanius, E-mail: wanius@if.sc.usp.br [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Travensolo, Regiane F. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Rodrigues, Nathalia C.; Muniz, João R. C. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Caruso, Célia S. [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Lemos, Eliana G. M. [Laboratório de Bioquímica de Microrganismos e de Plantas, Departamento de Tecnologia, UNESP, Jaboticabal (Brazil); Araujo, Ana Paula U. [Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Carrilho, Emanuel, E-mail: wanius@if.sc.usp.br [Grupo de Bioanalítica, Microfabricação e Separações, Instituto de Química de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil); Laboratório de Biofísica Molecular ‘Sérgio Mascarenhas’, Instituto de Física de São Carlos, Universidade de São Paulo (USP), São Carlos (Brazil)

    2008-02-01

    Glutathione S-transferase from X. fastidiosa (xfGST) has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.23 Å. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 Å, α = 63.45, β = 80.66, γ = 94.55°. xfGST crystals diffracted to 2.23 Å resolution on a rotating-anode X-ray source.

  8. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the mannose 6-phosphate isomerase from Salmonella typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Gowda, Giri; Sagurthi, Someswar Rao [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560 012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

    2008-02-01

    The cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of mannose 6-phosphate isomerase from S. typhimurium are reported. Mannose 6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible isomerization of d-mannose 6-phosphate (M6P) and d-fructose 6-phosphate (F6P). In the eukaryotes and prokaryotes investigated to date, the enzyme has been reported to play a crucial role in d-mannose metabolism and supply of the activated mannose donor guanosine diphosphate d-mannose (GDP-d-mannose). In the present study, MPI was cloned from Salmonella typhimurium, overexpressed in Escherichia coli and purified using Ni–NTA affinity column chromatography. Purified MPI crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 36.03, b = 92.2, c = 111.01 Å. A data set extending to 1.66 Å resolution was collected with 98.8% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. The asymmetric unit of the crystal cell was compatible with the presence of a monomer of MPI. A preliminary structure solution of the enzyme has been obtained by molecular replacement using Candida albicans MPI as the phasing model and the program Phaser. Further refinement and model building are in progress.

  9. Crystallization and preliminary X-ray diffraction analysis of West Nile virus

    International Nuclear Information System (INIS)

    Kaufmann, Bärbel; Plevka, Pavel; Kuhn, Richard J.; Rossmann, Michael G.

    2010-01-01

    Crystals of infectious West Nile virus were obtained and diffracted at best to about 25 Å resolution. Preliminary analysis of the diffraction pattern suggested tight hexagonal packing of the intact virus. West Nile virus, a human pathogen, is closely related to other medically important flaviviruses of global impact such as dengue virus. The infectious virus was purified from cell culture using polyethylene glycol (PEG) precipitation and density-gradient centrifugation. Thin amorphously shaped crystals of the lipid-enveloped virus were grown in quartz capillaries equilibrated by vapor diffusion. Crystal diffraction extended at best to a resolution of about 25 Å using synchrotron radiation. A preliminary analysis of the diffraction images indicated that the crystals had unit-cell parameters a ≃ b ≃ 480 Å, γ = 120°, suggesting a tight hexagonal packing of one virus particle per unit cell

  10. Crystallization and preliminary X-ray structural studies of a Melan-A pMHC–TCR complex

    International Nuclear Information System (INIS)

    Yuan, Fang; Georgiou, Theonie; Hillon, Theresa; Gostick, Emma; Price, David A.; Sewell, Andrew K.; Moysey, Ruth; Gavarret, Jessie; Vuidepot, Annelise; Sami, Malkit; Bell, John I.; Gao, George F.; Rizkallah, Pierre J.; Jakobsen, Bent K.

    2007-01-01

    A preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Melanocytes are specialized pigmented cells that are found in all healthy skin tissue. In certain individuals, diseased melanocytes can form malignant tumours, melanomas, which cause the majority of skin-cancer-related deaths. The melanoma-associated antigenic peptides are presented on cell surfaces via the class I major histocompatibility complex (MHC). Among the melanoma-associated antigens, the melanoma self-antigen A/melanoma antigen recognized by T cells (Melan-A/MART-1) has attracted attention because of its wide expression in primary and metastatic melanomas. Here, a preliminary X-ray crystal structural study of a soluble cognate T-cell receptor (TCR) in complex with a pMHC presenting the Melan-A peptide (ELAGIGILTV) is reported. The TCR and pMHC were refolded, purified and mixed together to form complexes, which were crystallized using the sitting-drop vapour-diffusion method. Single TCR–pMHC complex crystals were cryocooled and used for data collection. Diffraction data showed that these crystals belonged to space group P4 1 /P4 3 , with unit-cell parameters a = b = 120.4, c = 81.6 Å. A complete data set was collected to 3.1 Å and the structure is currently being analysed

  11. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-01-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygen

    Energy Technology Data Exchange (ETDEWEB)

    Geus, Daniël C. de, E-mail: d.de.geus@chem.leidenuniv.nl; Thomassen, Ellen A. J.; Feltz, Clarisse L. van der; Abrahams, Jan Pieter [Biophysical Structural Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden (Netherlands)

    2008-08-01

    Preliminary X-ray data collection and analysis for crystals of chlorite dismutase, a haem-based enzyme that very effectively reduces chlorite to chloride while producing molecular oxygen, is reported to 2.1 Å resolution. Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (< 2 kU mg{sup −1}) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P2{sub 1}2{sub 1}2 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79 Å. The crystals diffracted X-rays to 2.1 Å resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available.

  13. Crystallization and preliminary X-ray diffraction studies of the BTL2 lipase from the extremophilic microorganism Bacillus thermocatenulatus

    International Nuclear Information System (INIS)

    Carrasco-López, César; Godoy, Cesar; Rivas, Blanca de las; Fernández-Lorente, Gloria; Palomo, José M.; Guisán, José M.; Fernández-Lafuente, Roberto; Martínez-Ripoll, Martín; Hermoso, Juan A.

    2008-01-01

    BTL2, a thermostable enantioselective biocatalyst of interest for industrial applications, has been crystallized using the sitting-drop vapour-diffusion method. Preliminary X-ray data to 2.2 Å resolution allowed the determination of its unit-cell parameters and space group and initiation of its structure determination. Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that has been reported as an enantioselective biocatalyst for diverse reactions and that heads a group of enzymes that share high resistance towards many inactivation agents (heat, organic solvents, pH etc.). This makes BTL2 an important research target because of its potential industrial applications. BTL2 was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 13% MPD and 0.2 M ammonium acetate in 0.05 M sodium citrate pH 5.5–5.6. The crystals, which belonged to the orthorhombic space group I222 with unit-cell parameters a = 73.07, b = 129.08, c = 127.49 Å, allowed the collection of an X-ray data set to 2.2 Å resolution

  14. Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242

    International Nuclear Information System (INIS)

    Byrne-Steele, Miranda L.; Hughes, Ronny C.; Ng, Joseph D.

    2009-01-01

    The proliferating cell nuclear antigen (PCNA) from the eurypsychrophilic archaeon M. burtonii DSM 6242 has been cloned, overproduced, purified and crystallized. Crystals were deemed to be suitable for X-ray analysis and structure determination to 2.40 Å resolution. Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3 kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5 Å. A single MbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40 Å resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained

  15. Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

    International Nuclear Information System (INIS)

    Mathur, Divya; Anand, Kanchan; Mathur, Deepika; Jagadish, Nirmala; Suri, Anil; Garg, Lalit C.

    2007-01-01

    The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution. Phosphoglucose isomerase is a ubiquitous enzyme that catalyzes the isomerization of d-glucopyranose-6-phosphate to d-fructofuranose-6-phosphate. The present investigation reports the expression, purification, crystallization and preliminary crystallographic studies of the phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv, which shares 46% sequence identity with that of its human host. The recombinant protein, which was prepared using an Escherichia coli expression system, was crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group I2 1 2 1 2 1 , with unit-cell parameters a = 109.0, b = 119.8, c = 138.9 Å

  16. Purification, crystallization and preliminary X-ray crystallographic analysis of chitinase from Bacillus cereus NCTU2

    Energy Technology Data Exchange (ETDEWEB)

    Kuo, Chueh-Yuan [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Wu, Yue-Jin [Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010,Taiwan (China); Hsieh, Yin-Cheng; Guan, Hong-Hsiang [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013,Taiwan (China); Tsai, Huei-Ju [Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010,Taiwan (China); Lin, Yi-Hung; Huang, Yen-Chieh; Liu, Ming-Yih [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Li, Yaw-Kuen, E-mail: ykl@cc.nctu.edu.tw [Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010,Taiwan (China); Chen, Chun-Jung, E-mail: ykl@cc.nctu.edu.tw [Life Science Group, Research Division, National Synchrotron Radiation Research Center, Hsinchu 30076,Taiwan (China); Department of Physics, National Tsing-Hua University, Hsinchu 30013,Taiwan (China)

    2006-09-01

    The crystallization of B. cereus chitinase is reported. Chitinases (EC 3.2.1.14) are found in a broad range of organisms, including bacteria, fungi and higher plants, and play different roles depending on their origin. A chitinase from Bacillus cereus NCTU2 (ChiNCTU2) capable of hydrolyzing chitin as a carbon and nitrogen nutrient has been identified as a member of the family 18 glycoside hydrolases. ChiNCTU2 of molecular weight 36 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of chitinase crystals at 1.10 Å resolution, the crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.79, b = 48.79, c = 66.87 Å, β = 99.31°. Preliminary analysis indicates there is one chitinase molecule in the asymmetric unit, with a solvent content of 43.4%.

  17. Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase from Clostridium botulinum in the presence of its substrate pyruvate

    International Nuclear Information System (INIS)

    Atkinson, Sarah C.; Dobson, Renwick C. J.; Newman, Janet M.; Gorman, Michael A.; Dogovski, Con; Parker, Michael W.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyzes an important step in lysine biosynthesis. Here, the crystallization and preliminary diffraction analysis to 1.2 Å resolution of DHDPS from C. botulinum in the presence of its substrate pyruvate is reported. In this paper, the crystallization and preliminary X-ray diffraction analysis to near-atomic resolution of DHDPS from Clostridium botulinum crystallized in the presence of its substrate pyruvate are presented. The enzyme crystallized in a number of forms using a variety of PEG precipitants, with the best crystal diffracting to 1.2 Å resolution and belonging to space group C2, in contrast to the unbound form, which had trigonal symmetry. The unit-cell parameters were a = 143.4, b = 54.8, c = 94.3 Å, β = 126.3°. The crystal volume per protein weight (V M ) was 2.3 Å 3 Da −1 (based on the presence of two monomers in the asymmetric unit), with an estimated solvent content of 46%. The high-resolution structure of the pyruvate-bound form of C. botulinum DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  18. Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98

    International Nuclear Information System (INIS)

    Chauhan, Archana; Islam, Zeyaul; Jain, Rakesh Kumar; Karthikeyan, Subramanian

    2009-01-01

    Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported. Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively

  19. Crystallization and preliminary X-ray diffraction data of an LNA 7-mer duplex derived from a ricin aptamer

    International Nuclear Information System (INIS)

    Förster, Charlotte; Oberthuer, Dominik; Gao, Jiang; Eichert, André; Quast, Frederick G.; Betzel, Christian; Nitsche, Andreas; Erdmann, Volker A.; Fürste, Jens P.

    2009-01-01

    An all-LNA duplex was designed from the stem region of an RNA aptamer which has been generated against ricin. The LNA duplex was crystallized and preliminary X-ray diffraction analysis revealed diffraction to a resolution of up to 2.8 Å. Locked nucleic acids (LNAs) are modified nucleic acids which contain a modified sugar such as β-d-2′-O,4′-C methylene-bridged ribofuranose or other sugar derivatives in LNA analogues. The β-d-2′-O,4′-C methylene ribofuranose LNAs in particular possess high stability and melting temperatures, which makes them of interest for stabilizing the structure of different nucleic acids. Aptamers, which are DNAs or RNAs targeted against specific ligands, are candidates for substitution with LNAs in order to increase their stability. A 7-mer helix derived from the terminal part of an aptamer that was targeted against ricin was chosen. The ricin aptamer originally consisted of natural RNA building blocks and showed high affinity in ricin binding. For future stabilization of the aptamer, the terminal helix has been constructed as an ‘all-locked’ LNA and was successfully crystallized in order to investigate its structural properties. Optimization of crystal growth succeeded by the use of different metal salts as additives, such as CuCl 2 , MgCl 2 , MnCl 2 , CaCl 2 , CoCl 2 and ZnSO 4 . Preliminary X-ray diffraction data were collected and processed to 2.8 Å resolution. The LNA crystallized in space group P6 5 , with unit-cell parameters a = 50.11, b = 50.11, c = 40.72 Å. The crystals contained one LNA helix per asymmetric unit with a Matthews coefficient of 3.17 Å 3 Da −1 , which implies a solvent content of 70.15%

  20. Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium

    International Nuclear Information System (INIS)

    Azim, N.; Deery, E.; Warren, M. J.; Erskine, P.; Cooper, J. B.; Wood, S. P.; Akhtar, M.

    2013-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from B. megaterium was expressed and the enzyme was crystallized in a form which diffracts synchrotron radiation to high resolution. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution

  1. Expression, purification, crystallization and preliminary X-ray diffraction analysis of carbonyl reductase from Candida parapsilosis ATCC 7330

    International Nuclear Information System (INIS)

    Aggarwal, Nidhi; Mandal, P. K.; Gautham, Namasivayam; Chadha, Anju

    2013-01-01

    The expression, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies on C. parapsilosis carbonyl reductase are reported. The NAD(P)H-dependent carbonyl reductase from Candida parapsilosis ATCC 7330 catalyses the asymmetric reduction of ethyl 4-phenyl-2-oxobutanoate to ethyl (R)-4-phenyl-2-hydroxybutanoate, a precursor of angiotensin-converting enzyme inhibitors such as Cilazapril and Benazepril. The carbonyl reductase was expressed in Escherichia coli and purified by GST-affinity and size-exclusion chromatography. Crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.86 Å resolution. The asymmetric unit contained two molecules of carbonyl reductase, with a solvent content of 48%. The structure was solved by molecular replacement using cinnamyl alcohol dehydrogenase from Saccharomyces cerevisiae as a search model

  2. Crystallization and preliminary X-ray crystallographic studies of DnaJ from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Zhao, Shasha; Jin, Li; Niu, Siqiang; Yang, Wei; Zhang, Shaocheng; Guo, Zhen; Zhang, Hongpeng; Huang, Ailong; Yin, Yibing; Wang, Deqiang

    2013-01-01

    DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I2 1 2 1 2 1 and the diffraction resolution is 3.0 Å with unit-cell parameters a = 47.68, b = 104.45, c = 234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a V M value of 3.24 Å 3 Da −1 and a solvent content of 62.1%

  3. Purification, Crystallization, and Preliminary X-ray Analysis of Native Canavalin

    Science.gov (United States)

    Pusey, Marc; Dowell, Jennifer; Ng, Joseph; Gavira, Jose A.

    2003-01-01

    The protein canavalin is a 7S vicilin, from the Jack Bean, Canavalis ensfomis. Canavalin is described as a seed storage protein, an energy source for a developing seed, as no other known activity or function has been found. The protein was first isolated and crystallized by Sumner and Howell (J. Biol. Chem. 113, 607-610, 1936). Canavalin spontaneously crystallizes after proteolytic cleavage at neutral pH, which removes residues 1-46, 224-245, and 325-330 and produces peptides of approximately 25, 13, and 12 kDa. Preliminary gel filtration experiments indicated the presence of nucleic acid with the uncleaved protein. We developed a dual column procedure, ion exchange followed by hydroxy apatite chromatography, that effectively removes the nucleic acid and yields an essentially pure uncut canavalin preparation with an OD 280/260 ratio of approximately 1.9-2.0. Standard crystallization screens using this material gave a number of positive results having a common requirement for alcohols and Mg(2+) ion, with crystals typically appearing within a day or less. Optimization experiments to date have shown that we can obtain crystals from pH 6.5 to pH 8.2, using MPD from 5 to 20% and 0.05 to 0.2M Mg(2+) (sulfate or acetate). The crystals are of space group P2(sub 1)2(sub 1)2(sub 1), unit cell dimensions, and a complete data set to 1.5 Angstroms, resolution has now been collected at a synchrotron source. Most importantly, the crystals are not twinned, a persistent problem with the most commonly obtained rhombohedral form of proteolytically cleaved canavalin.

  4. Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

    Energy Technology Data Exchange (ETDEWEB)

    Ariza, Antonio; Vickers, Tim J.; Greig, Neil; Fairlamb, Alan H.; Bond, Charles S., E-mail: c.s.bond@dundee.ac.uk [Division of Biological Chemistry and Molecular Microbiology, Wellcome Trust Biocentre, School of Life Sciences, University of Dundee, Dundee DD1 5EH,Scotland (United Kingdom)

    2005-08-01

    The detoxification enzyme glyoxalase I from L. major has been crystallized. Preliminary molecular-replacement calculations indicate the presence of three glyoxalase I dimers in the asymmetric unit. Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

  5. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    International Nuclear Information System (INIS)

    Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2008-01-01

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution. Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 Å resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination

  6. Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms

    International Nuclear Information System (INIS)

    Reis, Marcelo Augusto dos; Saraiva, Antonio Marcos; Santos, Marcelo Leite dos; Souza, Anete Pereira de; Aparicio, Ricardo

    2012-01-01

    The crystallization and preliminary analysis of two crystal forms of survival protein E from X. fastidiosa are reported. The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, β = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, β = 94.76°

  7. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

    International Nuclear Information System (INIS)

    Jagadeesan, G.; Malathy, P.; Gunasekaran, K.; Harikrishna Etti, S.; Aravindhan, S.

    2014-01-01

    The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3 1 21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit

  8. Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

    International Nuclear Information System (INIS)

    Tonoli, Celisa Caldana Costa; Vieira, Plinio Salmazo; Ward, Richard John; Arni, Raghuvir Krishnaswamy; Oliveira, Arthur Henrique Cavalcante de; Murakami, Mario Tyago

    2009-01-01

    Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the nucleoside diphosphate kinase b from Leishmania major are reported. The crystals belonged to the trigonal space group P3 2 21 and diffracted to 2.18 Å resolution. Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host–parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 Å resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 Å. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3 2 21

  9. Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Jagadeesan, G. [Presidency College, Chennai 600 005 (India); Malathy, P.; Gunasekaran, K. [University of Madras, Chennai 600 025 (India); Harikrishna Etti, S. [GKM College of Engineering and Technology, Kamaraj Salai, Chennai 600 063 (India); Aravindhan, S., E-mail: aravindhanpresidency@gmail.com [Presidency College, Chennai 600 005 (India)

    2014-10-25

    The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3{sub 1}21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

  10. Crystallization and preliminary X-ray analysis of Na-SAA-2 from the human hookworm parasite Necator americanus

    International Nuclear Information System (INIS)

    Asojo, Oluwatoyin A.; Goud, Gaddam N.; Zhan, Bin; Ordonez, Katherine; Sedlacek, Meghan; Homma, Kohei; Deumic, Vehid; Gupta, Richi; Brelsford, Jill; Price, Merelyn K.; Ngamelue, Michelle N.; Hotez, Peter J.

    2010-01-01

    The purification, crystallization and preliminary X-ray diffraction analysis of a surface-associated antigen from the major human hookworm N. americanus is presented. Human hookworms are among the most pathogenic soil-transmitted helminths. These parasitic nematodes have co-evolved with the host and are able to maintain a high worm burden for decades without killing the human host. However, it is possible to develop vaccines against laboratory-challenge hookworm infections using either irradiated third-state infective larvae (L3) or enzymes from the adult parasites. In an effort to control hookworm infection globally, the Human Hookworm Vaccine Initiative, a product-development partnership with the Sabin Vaccine Institute to develop new control tools including vaccines, has identified a battery of protein antigens, including surface-associated antigens (SAAs) from L3. SAA proteins are characterized by a 13 kDa conserved domain of unknown function. SAA proteins are found on the surface of infective L3 stages (and some adult stages) of different nematode parasites, suggesting that they may play important roles in these organisms. The atomic structures and function of SAA proteins remain undetermined and in an effort to remedy this situation recombinant Na-SAA-2 from the most prevalent human hookworm parasite Necator americanus has been expressed, purified and crystallized. Useful X-ray data have been collected to 2.3 Å resolution from a crystal that belonged to the monoclinic space group C2 with unit-cell parameters a = 73.88, b = 35.58, c = 42.75 Å, β = 116.1°

  11. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex

    Energy Technology Data Exchange (ETDEWEB)

    Altenfeld, Anika; Wohlgemuth, Sabine [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Wehenkel, Annemarie [Institut Curie, CNRS UMR 3348/INSERM U1005, Bâtiment 110, Centre Universitaire, 91405 Orsay CEDEX (France); Vetter, Ingrid R. [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Musacchio, Andrea, E-mail: andrea.musacchio@mpi-dortmund.mpg.de [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); University of Duisburg-Essen, Universitätstrasse 1, 45141 Essen (Germany)

    2015-03-20

    The 800 kDa complex of the human Rod, Zwilch and ZW10 proteins (the RZZ complex) was reconstituted in insect cells, purified, crystallized and subjected to preliminary X-ray diffraction analysis. The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3{sub 1} (No. 144) or P3{sub 2} (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.

  12. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Gao, Xiong-Zhuo; Li, Lan-Fen; Su, Xiao-Dong; Zhao, XiaoJun; Liang, Yu-He

    2007-01-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°

  13. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiong-Zhuo [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Li, Lan-Fen; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, XiaoJun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

    2007-10-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.

  14. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    International Nuclear Information System (INIS)

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2012-01-01

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P6 5 , with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement

  15. Crystallization and preliminary X-ray analysis of AbsC, a novel regulator of antibiotic production in Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Stevenson, Clare E. M.; Kock, Holger; Mootien, Saraspadee; Davies, Sîan C.; Bibb, Mervyn J.; Lawson, David M.

    2007-01-01

    A novel regulator of antibiotic production in S. coelicolor, AbsC, has been crystallized in space group P2 1 2 1 2 1 . X-ray data to 2.25 Å resolution were collected on station PX 14.1 at Daresbury. Crystals of recombinant AbsC (subunit MW = 18 313 Da; 158 amino acids), a novel regulator of antibiotic production from Streptomyces coelicolor, were grown by vapour diffusion. The protein crystallizes in space group P2 1 2 1 2 1 , with unit-cell parameters a = 43.53, b = 121.30, c = 143.75 Å. Native data to a resolution of 2.25 Å were recorded at station PX 14.1 (Daresbury) from a single crystal. Preliminary analysis of these data suggests that the asymmetric unit contains four copies of the AbsC monomer, giving an estimated solvent content of 47.0%. AbsC belongs to the MarR family of proteins that mediate ligand-responsive transcriptional control

  16. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of bacterioferritin A from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Gupta, Vibha; Gupta, Rakesh K.; Khare, Garima; Salunke, Dinakar M.; Tyagi, Anil K.

    2008-01-01

    The cloning, purification and crystallization of a bacterioferritin from M. tuberculosis together with preliminary X-ray characterization of its crystals are reported. Bacterioferritins (Bfrs) comprise a subfamily of the ferritin superfamily of proteins that play an important role in bacterial iron storage and homeostasis. Bacterioferritins differ from ferritins in that they have additional noncovalently bound haem groups. To assess the physiological role of this subfamily of ferritins, a greater understanding of the structural details of bacterioferritins from various sources is required. The gene encoding bacterioferritin A (BfrA) from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein product was purified by affinity chromatography on a Strep-Tactin column and crystallized with sodium chloride as a precipitant at pH 8.0 using the vapour-diffusion technique. The crystals diffracted to 2.1 Å resolution and belonged to space group P4 2 , with unit-cell parameters a = 123.0, b = 123.0, c = 174.6 Å

  17. Crystallization and preliminary X-ray diffraction analysis of l,l-diaminopimelate aminotransferase (DapL) from Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Hudson, André O.; Girón, Irma; Dobson, Renwick C. J.

    2010-01-01

    A variant of the diaminopimelate/lysine pathway has recently been defined following the discovery of the enzyme l,l-diaminopimelate aminotransferase (DapL). The cloning of the cDNA, recombinant expression, purification and preliminary diffraction analysis of DapL from the alga C. reinhardtii are presented. In the anabolic synthesis of diaminopimelate and lysine in plants and in some bacteria, the enzyme l,l-diaminopimelate aminotransferase (DapL; EC 2.6.1.83) catalyzes the conversion of tetrahydrodipicolinic acid (THDPA) to l,l-diaminopimelate, bypassing the DapD, DapC and DapE enzymatic steps in the bacterial acyl pathways. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapL from the alga Chlamydomonas reinhardtii are presented. Protein crystals were grown in conditions containing 25%(w/v) PEG 3350 and 200 mM lithium sulfate and initially diffracted to ∼1.35 Å resolution. They belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 58.9, b = 91.8, c = 162.9 Å. The data were processed to 1.55 Å resolution with an R merge of 0.081, an R p.i.m. of 0.044, an R r.i.m of 0.093 and a V M of 2.28 Å 3 Da −1

  18. Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I

    International Nuclear Information System (INIS)

    Bogdanović, Xenia; Hesseler, Martin; Palm, Gottfried J.; Bornscheuer, Uwe T.; Hinrichs, Winfried

    2010-01-01

    The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported. DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2 1 2 1 2. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator

  19. Crystallization and preliminary X-ray crystallographic analysis of a new crystal form of hydroxylamine oxidoreductase from Nitrosomonas europaea

    International Nuclear Information System (INIS)

    Cedervall, Peder E.; Hooper, Alan B.; Wilmot, Carrie M.

    2009-01-01

    A new crystal form of N. europaea hydroxylamine oxidoreductase (space group P2 1 2 1 2) diffracted to 2.25 Å resolution at a third-generation synchrotron X-ray source. Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea is a homotrimeric protein that catalyzes the oxidation of hydroxylamine to nitrite. Each monomer, with a molecular weight of 67.1 kDa, contains seven c-type hemes and one heme P460, the porphyrin ring of which is covalently linked to a tyrosine residue from an adjacent subunit. HAO was first crystallized and structurally characterized at a resolution of 2.8 Å in 1997. The structure was solved in space group P6 3 and suffered from merohedral twinning. Here, a crystallization procedure is presented that yielded untwinned crystals belonging to space group P2 1 2 1 2, which diffracted to 2.25 Å resolution and contained one trimer in the asymmetric unit. The unit-cell parameters were a = 140.7, b = 142.6, c = 107.4 Å

  20. Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis

    International Nuclear Information System (INIS)

    Pereira-Rodríguez, Ángel; Fernández-Leiro, Rafael; González Siso, M. Isabel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

    2010-01-01

    β-Galactosidase from K. lactis has been expressed in S. cerevisiae, purified by affinity chromatography and crystallized in its native form. β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution

  1. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    Energy Technology Data Exchange (ETDEWEB)

    Maes, Dominique, E-mail: dominique.maes@vub.ac.be; Crabeel, Marjolaine [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium); Van de Weerdt, Cécile; Martial, Joseph [Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée de la Chimie 3, B-4000 Liège (Belgium); Peeters, Eveline; Charlier, Daniël [Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels (Belgium); Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium)

    2006-12-01

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.

  2. Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase.

    Science.gov (United States)

    Gunn, Natalie J; Gorman, Michael A; Dobson, Renwick C J; Parker, Michael W; Mulhern, Terrence D

    2011-03-01

    The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best crystals diffracted to ∼1.6 Å resolution. The crystals belonged to space group P2, with unit-cell parameters a=25.8, b=34.6, c=63.2 Å, β=99.4°.

  3. Crystallization and preliminary X-ray diffraction analysis of L,L-diaminopimelate aminotransferase (DapL) from Chlamydomonas reinhardtii.

    Science.gov (United States)

    Hudson, André O; Girón, Irma; Dobson, Renwick C J

    2011-01-01

    In the anabolic synthesis of diaminopimelate and lysine in plants and in some bacteria, the enzyme L,L-diaminopimelate aminotransferase (DapL; EC 2.6.1.83) catalyzes the conversion of tetrahydrodipicolinic acid (THDPA) to L,L-diaminopimelate, bypassing the DapD, DapC and DapE enzymatic steps in the bacterial acyl pathways. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapL from the alga Chlamydomonas reinhardtii are presented. Protein crystals were grown in conditions containing 25% (w/v) PEG 3350 and 200 mM lithium sulfate and initially diffracted to ∼1.35 Å resolution. They belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=58.9, b=91.8, c=162.9 Å. The data were processed to 1.55 Å resolution with an Rmerge of 0.081, an Rp.i.m. of 0.044, an Rr.i.m of 0.093 and a VM of 2.28 Å3 Da(-1).

  4. Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus

    International Nuclear Information System (INIS)

    Cho, Ching-Chang; Luo, Ching-Wei; Hsu, Chun-Hua

    2008-01-01

    PAT, an acetyltransferase from the archaeon S. solfataricus that specifically acetylates the chromatin protein Alba, was expressed, purified and crystallized. PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 Å resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 Å

  5. Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

    Energy Technology Data Exchange (ETDEWEB)

    Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation); Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S., E-mail: esipov@mx.ibch.ru [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics” (Russian Federation)

    2017-01-15

    Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°Ð¡, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

  6. Purification, crystallization and preliminary X-ray structure analysis of the banana lectin from Musa paradisiaca.

    Science.gov (United States)

    Singh, D D; Saikrishnan, K; Kumar, Prashant; Dauter, Z; Sekar, K; Surolia, A; Vijayan, M

    2004-11-01

    The banana lectin from Musa paradisiaca, MW 29.4 kDa, has been isolated, purified and crystallized. The trigonal crystals contain one dimeric molecule in the asymmetric unit. The structure has been solved using molecular replacement to a resolution of 3 A. The structure of the subunit is similar to that of jacalin-like lectins.

  7. Crystallization and preliminary X-ray analysis of vicenisaminyltransferase VinC

    International Nuclear Information System (INIS)

    Nango, Eriko; Minami, Atsushi; Kumasaka, Takashi; Eguchi, Tadashi

    2008-01-01

    The crystallization of VinC, a glycosyltransferase involved in the biosynthesis of the antitumour antibiotic vicenistatin, is reported. A recombinant glycosyltransferase, VinC, from Streptomyces halstedii HC34 has been crystallized at 293 K using PEG 3350 as precipitant. The diffraction pattern of the crystal extends to 2.0 Å resolution at 100 K using synchrotron radiation at SPring-8. The crystals are orthorhombic and belong to space group I222, with unit-cell parameters a = 98.21, b = 130.39, c = 140.11 Å. The presence of two molecules per asymmetric unit gives a crystal volume per protein weight (V M ) of 2.43 Å 3 Da −1 and a solvent content of 49.5% by volume

  8. Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

    Energy Technology Data Exchange (ETDEWEB)

    Saburi, Wataru; Hondoh, Hironori, E-mail: hondoh@abs.agr.hokudai.ac.jp [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan); Unno, Hideaki [Faculty of Engineering, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521 (Japan); Okuyama, Masayuki; Mori, Haruhide [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan); Nakada, Toshitaka [Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Matsuura, Yoshiki [Institute for Protein Research, Osaka University, Suita, Osaka 565-0871 (Japan); Kimura, Atsuo [Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589 (Japan)

    2007-09-01

    Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution. Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal.

  9. Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

    International Nuclear Information System (INIS)

    Saburi, Wataru; Hondoh, Hironori; Unno, Hideaki; Okuyama, Masayuki; Mori, Haruhide; Nakada, Toshitaka; Matsuura, Yoshiki; Kimura, Atsuo

    2007-01-01

    Dextran glucosidase from S. mutans was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.2 Å resolution. Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal α-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 Å. A native data set was collected to 2.2 Å resolution from a single crystal

  10. Crystallization and preliminary X-ray diffraction analysis of phosphoglycerate kinase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Bernardo-García, Noelia; Bartual, Sergio G.; Fulde, Marcus; Bergmann, Simone; Hermoso, Juan A.

    2011-01-01

    Phosphoglycerate kinase, a two-domain enzyme involved in the glycolytic pathway, has been crystallized. Both the N-terminal domain and a ternary complex of the full-length enzyme with substrates were crystallized by the sitting-drop vapour-diffusion method. Inclusion of the substrates was critical in order to obtain crystals of the full-length protein. Phosphoglycerate kinase (PGK) is a widespread two-domain enzyme that plays a critical role in the glycolytic pathway. Several glycolytic enzymes from streptococci have been identified as surface-exposed proteins that are involved in streptococcal virulence by their ability to bind host proteins. This binding allows pneumococcal cells to disseminate through the epithelial and endothelial layers. Crystallization of PGK from Streptococcus pneumoniae yielded orthorhombic crystals (space group I222, unit-cell parameters a = 62.73, b = 75.38, c = 83.63 Å). However, the unit cell of these crystals was not compatible with the presence of full-length PGK. Various analytical methods showed that only the N-terminal domain of PGK was present in the I222 crystals. The ternary complex of PGK with adenylyl imidodiphosphate (AMP-PNP) and 3-phospho-d-glycerate (3PGA) produced monoclinic crystals (space group P2 1 , unit-cell parameters a = 40.35, b = 78.23, c = 59.03 Å, β = 96.34°). Molecular replacement showed that this new crystal form contained full-length PGK, thereby indicating the relevance of including substrates in order to avoid proteolysis during the crystallization process

  11. Expression, purification, crystallization and preliminary X-ray analysis of the human NORE1 SARAH domain

    International Nuclear Information System (INIS)

    Kim, Hye Jin; Hwang, Eunha; Han, Young-Hyun; Choi, Saehae; Lee, Woo Cheol; Kim, Hye-Yeon; Jeon, Young Ho; Cheong, Chaejoon; Cheong, Hae-Kap

    2012-01-01

    The crystallization of the human NORE1 SARAH domain is reported. NORE1 is an important tumour suppressor in human cancers that interacts with the pro-apoptotic protein kinase MST1/2 through SARAH domains. The SARAH domain (residues 366–413) of human NORE1 was expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 2.7 Å resolution and belonged to space group P6 1 22, with unit-cell parameters a = b = 73.041, c = 66.092 Å, α = β = 90, γ = 120°

  12. Crystallization and preliminary X-ray diffraction analysis of mevalonate kinase from Methanosarcina mazei

    International Nuclear Information System (INIS)

    Zhuang, Ningning; Seo, Kyung Hye; Chen, Cong; Zhou, Jia; Kim, Seon Won; Lee, Kon Ho

    2012-01-01

    Recombinant mevalonate kinase from M. mazei has been crystallized. Diffraction data were collected to 2.08 Å resolution. Mevalonate kinase (MVK), which plays an important role in catalysing the biosynthesis of isoprenoid compounds derived from the mevalonate pathway, transforms mevalonate to 5-phosphomevalonate using ATP as a cofactor. Mevalonate kinase from Methanosarcina mazei (MmMVK) was expressed in Escherichia coli, purified and crystallized for structural analysis. Diffraction-quality crystals of MmMVK were obtained by the vapour-diffusion method using 0.32 M MgCl 2 , 0.08 M bis-tris pH 5.5, 16%(w/v) PEG 3350. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 97.11, b = 135.92, c = 46.03 Å. Diffraction data were collected to 2.08 Å resolution

  13. Crystallization and preliminary X-ray analysis of native and selenomethionyl vinorine synthase from Rauvolfia serpentina.

    Science.gov (United States)

    Ma, Xueyan; Koepke, Juergen; Bayer, Anja; Fritzsch, Günter; Michel, Hartmut; Stöckigt, Joachim

    2005-06-01

    Vinorine synthase (VS) is a central enzyme of the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases. So far, no three-dimensional structure with significant sequence homology with VS is known. Crystals of VS and selenomethionyl-labelled VS from the medicinal plant Rauvolfia serpentina have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants. VS crystals diffract to 2.8 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.3, b = 89.6, c = 136.2 A. The selenomethionyl VS crystal was nearly isomorphous with the VS crystal.

  14. Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH

    International Nuclear Information System (INIS)

    Raghunathan, Kannan; Vago, Frank S.; Ball, Terry; Yakubova, Nafissa; Grindem, David; Wedemeyer, William J.; Arvidson, Dennis N.

    2009-01-01

    Recombinant V. cholerae EpsH has been expressed, purified and crystallized. The crystals diffracted to 1.71 Å resolution. EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 Å. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V M of 2.1 Å 3 Da −1 , corresponding to 41.5% solvent content

  15. Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

    International Nuclear Information System (INIS)

    Nakajima, Yoshitaka; Ito, Kiyoshi; Xu, Yue; Yamada, Nozomi; Onohara, Yuko; Ito, Takashi; Yoshimoto, Tadashi

    2005-01-01

    P. gingivalis prolyl tripeptidyl aminopeptidase has been crystallized by the vapour-diffusion method. Diffraction data have been collected and processed to 2.1 Å resolution. A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6 3 22, with unit-cell parameters a = b = 149.4, c = 159.7 Å. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a V M value of 3.14 Å 3 Da −1 . Diffraction data were collected to 2.1 Å resolution using synchrotron radiation at the BL5 station of the Photon Factory

  16. Crystallization and preliminary X-ray characterization of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

    Energy Technology Data Exchange (ETDEWEB)

    Nakajima, Yoshitaka; Ito, Kiyoshi; Xu, Yue; Yamada, Nozomi; Onohara, Yuko; Ito, Takashi; Yoshimoto, Tadashi [Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521 (Japan)

    2005-12-01

    P. gingivalis prolyl tripeptidyl aminopeptidase has been crystallized by the vapour-diffusion method. Diffraction data have been collected and processed to 2.1 Å resolution. A recombinant form of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis has been crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as a precipitating agent. The crystals belong to the hexagonal space group P6{sub 3}22, with unit-cell parameters a = b = 149.4, c = 159.7 Å. The crystals are most likely to contain one subunit of a dimer in the asymmetric unit, with a V{sub M} value of 3.14 Å{sup 3} Da{sup −1}. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation at the BL5 station of the Photon Factory.

  17. Crystallization and preliminary X-ray diffraction studies of Murraya koenigii trypsin inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Shee, Chandan [Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667 (India); Singh, Tej P. [Department of Biophysics, All India Institute of Medical Sciences, New Delhi 100 029 (India); Kumar, Pravindra, E-mail: kumarfbs@iitr.ernet.in; Sharma, Ashwani K., E-mail: kumarfbs@iitr.ernet.in [Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667 (India)

    2007-04-01

    A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. A Kunitz-type trypsin inhibitor purified from the seeds of Murraya koenigii has been crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitating agent. The crystals belong to the tetragonal space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 75.8, c = 150.9 Å. The crystals contain two molecules in the asymmetric unit with a V{sub M} value of 2.5 Å{sup 3} Da{sup −1}. Diffraction was observed to 2.65 Å resolution and a complete data set was collected to 2.9 Å resolution.

  18. Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Xanthophyllomyces dendrorhous

    International Nuclear Information System (INIS)

    Polo, Aitana; Linde, Dolores; Estévez, Marta; Fernández-Lobato, María; Sanz-Aparicio, Julia

    2010-01-01

    The invertase from X. dendrorhous has been purified, deglycosylated and crystallized and diffraction data have been collected to 2.3 Å resolution. Xanthophyllomyces dendrorhous invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Its ability to produce neokestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which is highly glycosylated, failed to crystallize. Therefore, it was submitted to EndoH deglycosylating treatment and crystals were grown by vapour-diffusion methods. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 75.29, b = 204.93, c = 146.25 Å. Several diffraction data sets were collected using a synchrotron source. Self-rotation function and gel-filtration experiments suggested that the enzyme is a dimer with twofold symmetry

  19. Crystallization and preliminary X-ray analysis of d-2-hydroxyacid dehydrogenase from Haloferax mediterranei

    International Nuclear Information System (INIS)

    Domenech, J.; Baker, P. J.; Sedelnikova, S. E.; Rodgers, H. F.; Rice, D. W.; Ferrer, J.

    2009-01-01

    The d-2-hydroxyacid dehydrogenase from Haloferax mediterranei has been crystallized in two different forms. Diffraction data have been collected to 1.9 Å resolution for the non-productive ternary complex of the enzyme and to 2.7 Å for the selenomethionyl derivative. d-2-Hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with α-ketohexanoic acid and NAD + grew under these conditions. Crystals of form I diffracted to beyond 3.0 Å resolution and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 Å, β = 96.3°. Crystals of form II diffracted to beyond 2.0 Å resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 Å, α = 109.1, β = 107.5, γ = 95.9°. The calculated values for V M and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry

  20. Crystallization and preliminary X-ray crystallographic analysis of recombinant β-mannosidase from Aspergillus niger

    International Nuclear Information System (INIS)

    Demo, Gabriel; Fliedrová, Barbora; Weignerová, Lenka; Wimmerová, Michaela

    2013-01-01

    β-Mannosidase from Aspergillus niger was crystallized in the presence of d-mannose and the crystal diffracted to 2.41 Å resolution. The crystal belonged to the space group P1 with unit-cell parameters a= 62.37, b = 69.73, c = 69.90 Å, α = 108.20, β = 101.51, γ = 103.20°. β-Mannosidase (EC 3.2.1.25) is an important exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various oligomeric saccharide structures. β-Mannosidase from Aspergillus niger was expressed in Pichia pastoris and purified to clear homogeneity. β-Mannosidase was crystallized in the presence of d-mannose and the crystal diffracted to 2.41 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a = 62.37, b = 69.73, c = 69.90 Å, α = 108.20, β = 101.51, γ = 103.20°. The parameters derived from the data collection indicate the presence of one molecule in the asymmetric unit

  1. Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase

    International Nuclear Information System (INIS)

    Nyon, Mun Peak; Rice, David W.; Berrisford, John M.; Huang, Huazhang; Moir, Arthur J. G.; Craven, C. Jeremy; Nathan, Sheila; Mahadi, Nor Muhammad; Abu Bakar, Farah Diba

    2008-01-01

    Recombinant G. cingulata cutinase has been overexpressed and the protein has been purified and crystallized in the absence or presence of inhibitors. Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 Å resolution and belong to space group P4 1 2 1 2 or P4 3 2 1 2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2 1 2 1 2 1 and P2 1 , respectively, and diffract to approximately 2.5 Å resolution. All of the crystals are suitable for structural studies, which are currently ongoing

  2. Crystallization and preliminary X-ray studies of native and mutant intimin from enterohaemorrhagic Escherichia coli

    International Nuclear Information System (INIS)

    Yi, Yong; Gao, Feng; Mao, Xuhu; Xiao, Ming; Luo, Ping; Qi, Jianxun; Guo, Gang; Jing, Hua; Cui, Yan; Zou, Quanming

    2010-01-01

    Crystals of native intimin and its N916Y mutant from enterohaemorrhagic E. coli O157:H7 diffracted to 2.8 and 2.6 Å resolution, respectively. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a primarily food-borne bacterial pathogen that is capable of causing life-threatening human infections and poses a serious challenge to public health worldwide. The bacterial outer-membrane protein intimin plays a key role in the initiation process of EHEC infection. In this study, intimin from EHEC O157:H7 (Int188) and its N916Y mutant (IntN916Y) were purified and crystals of both were obtained using the hanging-drop vapour-diffusion method at 291 K. Data were collected from Int188 and IntN916Y crystals to 2.8 and 2.6 Å resolution, respectively. The crystal of Int188 belonged to the orthorhombic space group C2, with unit-cell parameters a = 235.16, b = 44.81, c = 129.12 Å, α = γ = 90, β = 97.53°. The crystal of IntN916Y belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 43.78, b = 92.49, c = 100.05 Å, α = β = γ = 90°

  3. Expression, purification, crystallization and preliminary X-ray analysis of tannase from Lactobacillus plantarum

    International Nuclear Information System (INIS)

    Wu, Mingbo; Peng, Xiaohong; Wen, Hua; Wang, Qin; Chen, Qianming; McKinstry, William J.; Ren, Bin

    2013-01-01

    A recombinant form of tannase from L. plantarum was purified and crystallized. A native data set has been collected to 1.65 Å resolution. Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase from Lactobacillus plantarum was cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space group P1, with unit-cell paramters a = 46.5, b = 62.8, c = 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution

  4. Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase.

    Science.gov (United States)

    Nyon, Mun Peak; Rice, David W; Berrisford, John M; Huang, Huazhang; Moir, Arthur J G; Craven, C Jeremy; Nathan, Sheila; Mahadi, Nor Muhammad; Abu Bakar, Farah Diba

    2008-06-01

    Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

  5. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    International Nuclear Information System (INIS)

    Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A.

    2009-01-01

    Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals. FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P2 1 3, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively

  6. Crystallization and preliminary X-ray analysis of Aspergillus oryzae catechol oxidase

    International Nuclear Information System (INIS)

    Kaljunen, Heidi; Gasparetti, Chiara; Kruus, Kristiina; Rouvinen, Juha; Hakulinen, Nina

    2011-01-01

    Catechol oxidase from A. oryzae was crystallized by the hanging-drop vapour-diffusion method. Catechol oxidase is an enzyme that catalyzes the oxidation of o-diphenols to the corresponding o-quinones. It is a copper-containing enzyme with a binuclear copper active site. Here, the crystallization and multiple-wavelength anomalous dispersion data collection of catechol oxidase from the mould fungus Aspergillus oryzae are described. During the purification, three forms of the enzyme (39.3, 40.5 and 44.3 kDa) were obtained. A mixture of these three forms was initially crystallized and gave crystals that diffracted to 2.5 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 118.9, c = 84.5 Å, α = β = 90, γ = 120°. A preparation containing only the shorter form (39.3 kDa) produced crystals that diffracted to 2.9 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.8, b = 95.3, c = 139.5 Å, α = β = γ = 90°

  7. Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase

    International Nuclear Information System (INIS)

    Kang, Gil Bu; Kim, Mun-Kyoung; Youn, Hyung-Seop; An, Jun Yop; Lee, Jung-Gyu; Park, Kyoung Ryoung; Lee, Sung Hang; Kim, Yongseong; Fukuoka, Shin-Ichi; Eom, Soo Hyun

    2010-01-01

    H. sapiens quinolinate phosphoribosyltransferase has been expressed, purified and crystallized. A diffraction data set has been collected and processed at 2.8 Å resolution. Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Å resolution. Native Hs-QPRTase crystals belonged to space group P2 1 , with unit-cell parameters a = 76.2, b = 137.1, c = 92.7 Å, β = 103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 Å 3 Da −1 , which corresponds to a solvent content of 49.9%

  8. Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX

    Energy Technology Data Exchange (ETDEWEB)

    Weadge, J.T.; Robinson, H.; Yip, P. P.; Arnett, K.; Tipton, P. A.; Howell, P. L.

    2010-05-01

    AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 {angstrom}, {beta} = 95.7{sup o}. The crystals exhibited the symmetry of space group P2{sub 1} and diffracted to a minimum d-spacing of 2.1 {angstrom}. On the basis of the Matthews coefficient (V{sub M} = 2.25 {angstrom}{sup 3} Da{sup -1}), two molecules were estimated to be present in the asymmetric unit.

  9. Cloning, expression, crystallization and preliminary X-ray characterization of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus

    International Nuclear Information System (INIS)

    Ichiki, Shin-ichi; Nakamura, Shota; Ohkubo, Tadayasu; Kobayashi, Yuji; Hasegawa, Jun; Uchiyama, Susumu; Nishihara, Hirofumi; Mizuta, Keiko; Sambongi, Yoshihiro

    2005-01-01

    Cytochrome c 552 of a moderate thermophile, H. thermoluteolus, was overexpressed in E. coli and crystallized for X-ray diffraction study. The amino-acid sequence of cytochrome c 552 (PH c 552 ) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c 552 ), and from a mesophile, Pseudomonas aeruginosa (PA c 551 ). The PH c 552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c 552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222 1 , with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 Å. The crystals diffract X-rays to around 2.1 Å resolution

  10. Crystallization and preliminary X-ray diffraction study of recombinant ribokinase from Thermus Species 2.9

    Energy Technology Data Exchange (ETDEWEB)

    Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” (Russian Federation); Muravieva, T. I.; Esipov, R. S., E-mail: espiov@mx.ibch.ru [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P., E-mail: inna@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” (Russian Federation)

    2016-11-15

    Ribokinase from a thermophilic strain of Thermus species 2.9 belonging to the carbohydrate ribokinase family (EC 2.7.1.15) was isolated, purified, and crystallized. The crystallization conditions were found by the vapor-diffusion technique and were then optimized to apply the capillary counter-diffusion technique. The X-ray diffraction data set was collected from the crystals, which were grown by the counter-diffusion technique, at the SPring-8 synchrotron radiation facility to 2.87 Å resolution. The crystals belong to sp. gr. P12{sub 1}1 and have the following unit-cell parameters: a = 81.613 Å, b = 156.132 Å, c = 87.714 Å, α = γ = 90°, β = 103.819°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the protein by the molecular-replacement method.

  11. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    International Nuclear Information System (INIS)

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2 1 2 1 2 1 . X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2 1 2 1 2 1 and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research

  12. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    Energy Technology Data Exchange (ETDEWEB)

    Wood, S. J. [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Li, X.-L.; Cotta, M. A. [Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois 61604 (United States); Biely, P. [Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava (Slovakia); Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R., E-mail: rajp@anl.gov [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2008-04-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  13. Purification, crystallization and preliminary X-ray diffraction analysis of a plant subtilase

    International Nuclear Information System (INIS)

    Rose, Rolf; Huttenlocher, Franziska; Cedzich, Anna; Kaiser, Markus; Schaller, Andreas; Ottmann, Christian

    2009-01-01

    The first crystallographic study of a plant subtilase, SBT3 from S. lycopersicum, is reported. The subtilase SBT3 from Solanum lycopersicum (tomato) was purified from a tomato cell culture and crystallized using the sitting-drop vapour-diffusion method. A native data set was collected to 2.5 Å resolution at 100 K using synchrotron radiation. For experimental phasing, CsCl-derivative and tetrakis(acetoxymercuri)methane (TAMM) derivative crystals were employed for MIRAS phasing. Three caesium sites and one TAMM site were identified, which allowed solution of the structure

  14. Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Paul T.; Raghunathan, Kannan; Spurbeck, Rachel R.; Arvidson, Cindy G.; Arvidson, Dennis N. (MSU)

    2010-09-02

    Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 {angstrom} resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 {angstrom}. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V{sub M} of 2.8 {angstrom}{sup 3} Da{sup -1}, corresponding to 55.2% solvent content.

  15. Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-[beta]-lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, N.; Toney, J.H.; Fitzgerald, P.M.D. (Merck)

    2010-07-20

    The CphA metallo-{beta}-lactamase from Aeromonas hydrophilia has been expressed, purified and crystallized by the hanging-drop vapor-diffusion method using ammonium sulfate as the precipitant. The crystals exhibit orthorhombic symmetry (P2{sub 1}2{sub 1}2), with unit-cell parameters a = 40.75, b = 42.05, c = 128.88 {angstrom}. There is one monomer in the asymmetric unit and the solvent content is estimated to be 44% by volume. A data set extending to 1.8 {angstrom} has been measured.

  16. Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia)

    International Nuclear Information System (INIS)

    Sathya Moorthy, Pon.; Neelagandan, K.; Balasubramanian, M.; Ponnuswamy, M. N.

    2009-01-01

    Crystallization of pigeon haemoglobin at low pH (5.5) and high ionic concentration (1 M) using the hanging-drop vapour-diffusion method is reported. Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°

  17. Crystallization and preliminary X-ray analysis of carnein, a serine protease from Ipomoea carnea.

    NARCIS (Netherlands)

    Patel, A.K.; Oosterwijk, N. van; Singh, V.K.; Rozeboom, H.J.; Kalk, K.H.; Siezen, R.J.; Jagannadham, M.V.; Dijkstra, B.W.

    2009-01-01

    Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of grass carp β2-microglobulin

    International Nuclear Information System (INIS)

    Chen, Weihong; Chu, Fuliang; Peng, Hao; Zhang, Jianhua; Qi, Jianxun; Jiang, Fan; Xia, Chun; Gao, Feng

    2008-01-01

    Grass carp β 2 -microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. β 2 -Microglobulin (β 2 m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β 2 m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β 2 m (Ctid-β 2 m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da −1 and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β 2 m as a model

  19. Crystallization and preliminary X-ray crystallographic studies of O-methyltransferase from Anabaena PCC 7120

    International Nuclear Information System (INIS)

    Li, Guoming; Tang, Zhenting; Meng, Geng; Dai, Kesheng; Zhao, Jindong; Zheng, Xiaofeng

    2009-01-01

    The O-methyltransferase (OMT) from the Anabaena PCC 7120 has been overexpressed in a soluble form in E. coli, purified and crystallized. The crystals belonged to space group C222 1 and diffracted to 2.4 Å resolution. O-Methyltransferase (OMT) is a ubiquitous enzyme that exists in bacteria, plants and humans and catalyzes a methyl-transfer reaction using S-adenosyl-l-methionine as a methyl donor and a wide range of phenolics as acceptors. To investigate the structure and function of OMTs, omt from Anabaena PCC 7120 was cloned into expression vector pET21a and expressed in a soluble form in Escherichia coli strain BL21 (DE3). The recombinant OMT protein was purified to homogeneity using a two-step strategy. Crystals of OMT that diffracted to a resolution of 2.4 Å were obtained using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222 1 , with unit-cell parameters a = 131.620, b = 227.994, c = 150.777 Å, α = β = γ = 90°. There are eight molecules per asymmetric unit

  20. Crystallization and preliminary X-ray study of the common edible mushroom (Agaricus bisporus) lectin.

    Science.gov (United States)

    Carrizo, Maria E; Irazoqui, Fernando J; Lardone, Ricardo D; Nores, Gustavo A; Curtino, Juan A; Capaldi, Stefano; Perduca, Massimiliano; Monaco, Hugo L

    2004-04-01

    The lectin from the common edible mushroom Agaricus bisporus (ABL) belongs to the group of proteins that have the property of binding the Thomsen-Friedenreich antigen (T-antigen) selectively and with high affinity, but does not show any sequence similarity to the other proteins that share this property. The ABL sequence is instead similar to those of members of the saline-soluble fungal lectins, a protein family with pesticidal properties. The presence of different isoforms has been reported. It has been found that in order to be able to grow diffraction-quality crystals of the lectin, it is essential to separate the isoforms, which was performed by preparative isoelectric focusing. Using standard procedures, it was possible to crystallize the most basic of the forms by either vapour diffusion or equilibrium dialysis, but attempts to grow crystals of the other more acidic forms were unsuccessful. The ABL crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 93.06, b = 98.16, c = 76.38 A, and diffract to a resolution of 2.2 A on a conventional source at room temperature. It is expected that the solution of this structure will yield further valuable information on the differences in the T-antigen-binding folds and will perhaps help to clarify the details of the ligand binding to the protein.

  1. Crystallization and preliminary X-ray analysis of CooA from Carboxydothermus hydrogenoformans

    International Nuclear Information System (INIS)

    Komori, Hirofumi; Satomoto, Kensuke; Ueda, Yasufumi; Shibata, Naoki; Inagaki, Sayaka; Yoshioka, Shiro; Aono, Shigetoshi; Higuchi, Yoshiki

    2006-01-01

    CooA from C. hydrogenoformans has been crystallized by the vapour-diffusion method using polyethylene glycol as a precipitant. The crystal diffracted to 2.3 Å resolution. CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism. CooA from Carboxydothermus hydrogenoformans has been overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal belongs to space group P2 1 , with unit-cell parameters a = 61.8, b = 94.7, c = 92.8 Å, β = 104.8°. The native and anomalous difference Patterson maps indicated that two CooA dimers are contained in the asymmetric unit and are related by a translational symmetry almost parallel to the c axis

  2. Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin

    International Nuclear Information System (INIS)

    Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

    2012-01-01

    A crystal of nurse shark β 2 -microglobulin diffracted to 2.3 Å resolution and belonged to space group P3 2 21, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å 3 Da −1 , corresponding to 62.5% solvent content. β 2 -Microglobulin (β 2 m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide–MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β 2 m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β 2 m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3 2 21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β 2 m and IgSF in the AIS

  3. Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

    International Nuclear Information System (INIS)

    Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre

    2006-01-01

    Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4 1 32 or P4 3 32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å

  4. Purification, crystallization and preliminary X-ray diffraction of fluorescence recovery protein from Synechocystis PCC 6803

    International Nuclear Information System (INIS)

    Liu, Ting; Shuai, Yingli; Zhou, Honggang

    2011-01-01

    Fluorescence recovery protein from Synechocystis PCC 6803 plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. The full-length form and a truncated form were overexpressed, purified and crystallized, and diffraction was observed to 2.75 Å resolution. Fluorescence recovery protein (FRP), which is encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. As the crystal structure of FRP may provide information about the biological functions and mechanism of action of the protein, recombinant full-length FRP and a truncated form were overexpressed, purified and crystallized at 291 K using ethylene imine polymer as the precipitant. An FRP data set was collected to a resolution of 2.75 Å at low temperature (100 K). The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 61.9, c = 160.7 Å, α = β = γ = 90°. Assuming that the asymmetric unit contains three molecules, the Matthews coefficient was calculated to be 2.1 Å 3 Da −1

  5. Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase

    NARCIS (Netherlands)

    Verdon, Grégory; Albers, Sonja-V.; Dijkstra, Bauke W.; Driessen, Arnold J.M.; Thunnissen, Andy-Mark W.H.

    2002-01-01

    In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding

  6. Crystallization and preliminary X-ray diffraction studies of the pneumococcal teichoic acid phosphorylcholine esterase Pce

    Energy Technology Data Exchange (ETDEWEB)

    Lagartera, Laura; González, Ana; Stelter, Meike; García, Pedro; Kahn, Richard; Menéndez, Margarita; Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es

    2005-02-01

    The modular choline-binding protein Pce, the phosphorylcholine esterase from S. pneumoniae, has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a derivative with a gadolinium complex has been collected to 2.7 Å resolution.

  7. Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre, E-mail: marie-pierre.egloff@afmb.univ-mrs.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2006-04-01

    Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4{sub 1}32 or P4{sub 3}32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.

  8. Complex assembly, crystallization and preliminary X-ray crystallographic studies of duck MHC class I molecule

    International Nuclear Information System (INIS)

    Zhang, Jianhua; Chen, Yong; Gao, Feng; Chen, Weihong; Qi, Jianxun; Xia, Chun

    2009-01-01

    Using a peptide derived from H5N1, a complex of duck MHC class I molecule (DuMHC I) with duck β 2 -microglobulin (Duβ 2 m) was assembled and crystallized. Initial structure analysis indicated that the crystals did not contain the complete DuMHC I complex but instead contained DuMHC I α3-domain and Duβ 2 m subunits. In order to understand the biological properties of the immune systems of waterfowl and to establish a system for structural studies of duck class I major histocompatibility complex (DuMHC I), a complex of DuMHC I with duck β 2 -microglobulin (Duβ 2 m) and the peptide AEIEDLIF (AF8) derived from H5N1 NP residues 251–258 was assembled. The complex was crystallized; the crystals belonged to space group C222 1 , with unit-cell parameters a = 54.7, b = 72.4, c = 102.2 Å, and diffracted to 2.3 Å resolution. Matthews coefficient calculation and initial structure determination by molecular replacement showed that the crystals did not contain the whole DuMHC I complex, but instead contained the DuMHC I α3 domain and a Duβ2m molecule (DuMHC I α3+β2m). Another complex of DuMHC I with the peptide IDWFDGKE derived from a chicken fusion protein also generated the same results. The stable structure of DuMHC I α3+β2m may reflect some unique characteristics of DuMHC I and pave the way for novel MHC structure-related studies in the future

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutSβ

    International Nuclear Information System (INIS)

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.; Iyer, Ravi R.; Changela, Anita; Modrich, Paul L.; Beese, Lorena S.

    2011-01-01

    Human MutSβ is a 232 kDa heterodimer (MSH2–MSH3) involved in the lesion-recognition step of mismatch repair. Here, the overexpression, purification, biochemical characterization and cocrystallization of MutSβ with a duplex DNA substrate are reported. MutSβ is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutSα (MSH2–MSH6). Although mismatch recognition by MutSα has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutSβ. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutSβ and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported

  10. Purification, crystallization and preliminary X-ray diffraction of human S100A15

    Energy Technology Data Exchange (ETDEWEB)

    Boeshans, Karen M. [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States); Wolf, Ronald; Voscopoulos, Christopher [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Gillette, William; Esposito, Dominic [Protein Expression Laboratory, Research Technology Program, National Cancer Institute, SAIC-Frederick Inc., Frederick, MD 21702 (United States); Mueser, Timothy C. [Department of Chemistry, University of Toledo, Toledo, OH 43606 (United States); Yuspa, Stuart H. [Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Ahvazi, Bijan, E-mail: ahvazib@mail.nih.gov [X-ray Crystallography Facility, NIAMS, National Institutes of Health, Bethesda, MD 20892 (United States)

    2006-05-01

    S100 proteins are differentially expressed during epithelial cell maturation, tumorigenesis and inflammation. The novel human S100A15 protein has been cloned, expressed, purified and crystallized in two crystal forms, a triclinic and a monoclinic form, which diffract to 1.7 and 2.0 Å, respectively. Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 Å, α = 71.2, β = 68.1, γ = 67.8° and an estimated two molecules in the asymmetric unit, and diffract to 1.7 Å resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 Å, β = 128.2° and an estimated one molecule in the asymmetric unit, and diffract to 2.0 Å resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.

  11. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-01-01

    Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

  12. Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin

    OpenAIRE

    Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

    2012-01-01

    A crystal of nurse shark β2-microglobulin diffracted to 2.3 Å resolution and belonged to space group P3221, with unit-cell parameters a = b = 88.230, c = 67.146 Å and two molecules per asymmetric unit. The Matthews coefficient V M was calculated to be about 3.28 Å3 Da−1, corresponding to 62.5% solvent content.

  13. Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.

    Science.gov (United States)

    Kellosalo, Juho; Kajander, Tommi; Honkanen, Riina; Goldman, Adrian

    2013-02-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.

  14. Expression, purification, crystallization and preliminary X-ray diffraction analysis of nurse shark β2-microglobulin.

    Science.gov (United States)

    Lu, Shuangshuang; Yao, Shugang; Chen, Rong; Zhang, Nianzhi; Chen, Jianmin; Gao, Feng; Xia, Chun

    2012-04-01

    β(2)-Microglobulin (β(2)m) is an essential subunit of the major histocompatibility complex (MHC) class I molecule that helps to stabilize the structure of peptide-MHC I (pMHC I). It is also one of the typical immunoglobulin superfamily (IgSF) molecules in the adaptive immune system (AIS). Sharks belong to the cartilaginous fish, which are the oldest jawed vertebrate ancestors with an AIS to exist in the world. Thus, the study of cartilaginous fish β(2)m would help in understanding the evolution of IgSF molecules. In order to demonstrate this, β(2)m from a cartilaginous fish, nurse shark (Ginglymostoma cirratum), was expressed, refolded, purified and crystallized. Diffraction data were collected to a resolution of 2.3 Å. The crystal belonged to space group P3(2)21, with unit-cell parameters a = b = 88.230, c = 67.146 Å. The crystal structure contained two molecules in the asymmetric unit. The results will provide structural information for study of the evolution of β(2)m and IgSF in the AIS. © 2012 International Union of Crystallography. All rights reserved.

  15. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  16. Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein

    International Nuclear Information System (INIS)

    Bai, Xiaoyun; Meng, Geng; Li, Guoming; Luo, Ming; Zheng, Xiaofeng

    2009-01-01

    A C-terminal truncation construct of human PACSIN 1 (1–344) has been purified and crystallized. Diffraction data were collected to 3.0 Å resolution. PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1–344) was crystallized and diffracted to a resolution of 3.0 Å. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 Å, α = 90.00, β = 113.61, γ = 90.00°. There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%

  17. Crystallization and preliminary X-ray crystallographic studies of human FAIM protein

    International Nuclear Information System (INIS)

    Li, Guoming; Qu, Linglong; Meng, Geng; Bai, Xiaoyun; Dai, Kesheng; Zheng, Xiaofeng

    2010-01-01

    Full-length human Fas apoptosis inhibitory molecule (FAIM) and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1–90) was crystallized and diffracted to a resolution of 2.5 Å. Fas apoptosis inhibitory molecule (FAIM), an antagonist of Fas-induced cell death, is highly conserved and is broadly expressed in many tissues. It has been found that FAIM can stimulate neurite outgrowth in PC12 cells and primary neurons. However, the molecular mechanisms of action of FAIM are not understood in detail. Here, full-length human FAIM and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1–90) was crystallized and diffracted to a resolution of 2.5 Å; the crystal belonged to space group P3 1 , with unit-cell parameters a = b = 58.02, c = 71.11 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit

  18. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    International Nuclear Information System (INIS)

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-01-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit

  19. Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia)

    Science.gov (United States)

    Sathya Moorthy, Pon.; Neelagandan, K.; Balasubramanian, M.; Ponnuswamy, M. N.

    2009-01-01

    Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°. PMID:19194000

  20. Crystallization and preliminary X-ray diffraction studies of l-rhamnose isomerase from Pseudomonas stutzeri

    International Nuclear Information System (INIS)

    Yoshida, Hiromi; Wayoon, Poonperm; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2006-01-01

    Recombinant l-rhamnose isomerase from P. stutzeri has been crystallized. Diffraction data have been collected to 2.0 Å resolution. l-Rhamnose isomerase from Pseudomonas stutzeri (P. stutzeril-RhI) catalyzes not only the reversible isomerization of l-rhamnose to l-rhamnulose, but also isomerization between various rare aldoses and ketoses. Purified His-tagged P. stutzeril-RhI was crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 74.3, b = 104.0, c = 107.0 Å, β = 106.8°. Diffraction data have been collected to 2.0 Å resolution. The molecular weight of the purified P. stutzeril-RhI with a His tag at the C-terminus was confirmed to be 47.7 kDa by MALDI–TOF mass-spectrometric analysis and the asymmetric unit is expected to contain four molecules

  1. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    Energy Technology Data Exchange (ETDEWEB)

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul, E-mail: hnc@pusan.ac.kr [College of Pharmacy and Research Institute for Drug Development, Pusan National University, Jangjeon-dong, Geumjeong-gu, Busan 609-735 (Korea, Republic of)

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  2. Purification, crystallization and preliminary X-ray crystallographic analysis of PBP4 from Listeria monocytogenes

    International Nuclear Information System (INIS)

    Jeong, Jae-Hee; Bae, Ji-Eun; Kim, Yeon-Gil

    2011-01-01

    The crystallization of PBP4 from L. monocytogenes is reported. Penicillin-binding proteins (PBPs), which catalyze peptidoglycan synthesis, have been extensively studied as a well established target of antimicrobial agents, including β-lactam derivatives. However, remarkable resistance to β-lactams has developed among pathogenic bacteria since the clinical use of penicillin began. Recently, the glycosyltransferase (GT) domain of class A PBPs has been proposed as an attractive target for antibiotic development as moenomycin-bound GT-domain structures have been determined. In this study, a class A PBP4 from Listeria monocytogenes was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 84.6, b = 127.8, c = 54.9 Å. The structural information will contribute to the further development of moenomycin-derived antibiotics possessing broad-spectrum activity

  3. Purification, crystallization and preliminary X-ray analysis of a hexameric β-glucosidase from wheat

    International Nuclear Information System (INIS)

    Sue, Masayuki; Yamazaki, Kana; Kouyama, Jun-ichi; Sasaki, Yasuyuki; Ohsawa, Kanju; Miyamoto, Toru; Iwamura, Hajime; Yajima, Shunsuke

    2005-01-01

    Recombinant β-glucosidase from wheat seedlings complexed with a substrate aglycone has been crystallized in a hexameric active form. A diffraction data set has been collected at 1.7 Å. The wheat β-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6×His at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO 4 and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 Å at the Photon Factory. The crystal belongs to space group P4 1 32, with unit-cell parameters a = b = c = 194.65 Å, α = β = γ = 90°. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%

  4. Purification, crystallization and preliminary x-ray crystallographic studies of RAIDD Death-Domain (DD).

    Science.gov (United States)

    Jang, Tae-ho; Park, Hyun Ho

    2009-06-03

    Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

  5. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  6. Expression, Purification, Crystallization and Preliminary X-ray Analysis of Pseudomonas fluorescens AlgK

    Energy Technology Data Exchange (ETDEWEB)

    Keiski,C.; Yip, P.; Robinson, H.; Burrows, L.; Howell, P.

    2007-01-01

    AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15 {angstrom}, = 96.97{sup o}. The crystals exhibit the symmetry of space group P2{sub 1} and diffract to a minimum d-spacing of 2.5 {angstrom} at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (V{sub M} = 2.53 {angstrom}{sup 3} Da{sup -1}), four protein molecules are estimated to be present in the asymmetric unit.

  7. Crystallization and preliminary X-ray analysis of the V domain of human nectin-2

    International Nuclear Information System (INIS)

    Qian, Xiaomin; Qi, Jianxun; Chu, Fuliang; Liu, Jun; Li, Qing; Yan, Jinghua

    2009-01-01

    Crystals of the V domain of human nectin-2 diffracted to 1.85 Å resolution and were monoclinic, belonging to space group P2 1 , with unit-cell parameters a = 52.3, b = 43.9, c = 56.1 Å, β = 118.2°. Nectin-2 belongs to a family of immunoglobulin-like cell adhesion molecules that are characterized by the presence of three immunoglobulin-like domains (V, C2 and C2) in the extracellular region. The V domain plays important roles in cell adhesion, NK cell activation and the entry of some herpesvirus. In this study, the V domain of human nectin-2 was expressed in Escherichia coli in the form of inclusion bodies, which were subsequently denatured and refolded. The soluble protein was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 1.85 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 52.3, b = 43.9, c = 56.1 Å, β = 118.2°

  8. Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD

    Directory of Open Access Journals (Sweden)

    Hyun Ho Park

    2009-06-01

    Full Text Available Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD and a C-terminal Death-Domain (DD. Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3121 (or its enantiomorph P3221 with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 Å and γ = 120°. The crystals were obtained at room temperature and diffracted to 2.0 Å resolution.

  9. Overexpression, purification, crystallization and preliminary X-ray cystallographic studies of a proline-specific aminopeptidase from Aneurinibacillus sp. strain AM-1

    International Nuclear Information System (INIS)

    Akioka, Makoto; Nakano, Hiroaki; Horikiri, Aya; Tsujimoto, Yoshiyuki; Matsui, Hiroshi; Shimizu, Tetsuya; Nakatsu, Toru; Kato, Hiroaki; Watanabe, Kunihiko

    2006-01-01

    Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out. To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination

  10. Crystallization and preliminary X-ray diffraction analysis of nepenthesin-1

    Czech Academy of Sciences Publication Activity Database

    Fejfarová, Karla; Man, Petr; Mrázek, Hynek; Kádek, Alan; Dušková, Jarmila; Skálová, Tereza; Kolenko, Petr; Koval, Tomáš; Stránský, Jan; Hašek, Jindřich; Dohnálek, Jan

    2014-01-01

    Roč. 21, č. 1 (2014), s. 32 ISSN 1211-5894. [Discussions in Structural Molecular Biology /12./. 13.03.2014-15.03.2014, Nové Hrady] R&D Projects: GA MŠk EE2.3.30.0029; GA MŠk LG14009; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:61388971 ; RVO:86652036 Keywords : plant aspartate protease * crystallization Subject RIV: CE - Biochemistry; EI - Biotechnology ; Bionics (MBU-M); EB - Genetics ; Molecular Biology (BTO-N)

  11. Crystallization and preliminary X-ray analysis of an exotype alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, a member of polysaccharide lyase family 15

    International Nuclear Information System (INIS)

    Ochiai, Akihito; Yamasaki, Masayuki; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2006-01-01

    The crystallization and preliminary X-ray characterization of a family PL-15 exotype alginate lyase are presented. Almost all alginate lyases depolymerize alginate in an endolytical fashion via a β-elimination reaction. The alginate lyase Atu3025 from Agrobacterium tumefaciens strain C58, consisting of 776 amino-acid residues, is a novel exotype alginate lyase classified into polysaccharide lyase family 15. The enzyme was crystallized at 293 K by sitting-drop vapour diffusion with polyethylene glycol 4000 as a precipitant. Preliminary X-ray analysis showed that the Atu3025 crystal belonged to space group P2 1 and diffracted to 2.8 Å resolution, with unit-cell parameters a = 107.7, b = 108.3, c = 149.5 Å, β = 91.5°

  12. Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036

    International Nuclear Information System (INIS)

    Tishchenko, Svetlana; Gabdulkhakov, Azat; Tin, Uliana; Kostareva, Olga; Lin, Chen; Katanaev, Vladimir L.

    2012-01-01

    D. melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization and preliminary X-ray crystallographic analysis of the complex of the two proteins are reported. Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source

  13. Crystallization and preliminary X-ray analysis of the inducible lysine decarboxylase from Escherichia coli

    International Nuclear Information System (INIS)

    Alexopoulos, Eftichia; Kanjee, Usheer; Snider, Jamie; Houry, Walid A.; Pai, Emil F.

    2008-01-01

    The structure of the decameric inducible lysine decarboxylase from E. coli was determined by SIRAS using a hexatantalum dodecabromide (Ta 6 Br 12 2+ ) derivative. Model building and refinement are under way. The decameric inducible lysine decarboxylase (LdcI) from Escherichia coli has been crystallized in space groups C2 and C222 1 ; the Ta 6 Br 12 2+ cluster was used to derivatize the C2 crystals. The method of single isomorphous replacement with anomalous scattering (SIRAS) as implemented in SHELXD was used to solve the Ta 6 Br 12 2+ -derivatized structure to 5 Å resolution. Many of the Ta 6 Br 12 2+ -binding sites had twofold and fivefold noncrystallographic symmetry. Taking advantage of this feature, phase modification was performed in DM. The electron-density map of LdcI displays many features in agreement with the low-resolution negative-stain electron-density map [Snider et al. (2006 ▶), J. Biol. Chem.281, 1532–1546

  14. Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli

    International Nuclear Information System (INIS)

    Zheng, Jimin; Lee, Daniel C.; Jia, Zongchao

    2009-01-01

    Isocitrate dehydrogenase kinase/phosphatase has been crystallized in three different crystal forms. Data were collected from each crystal form for structure determination. The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P4 1 2 1 2, P3 2 21 and P2 1 2 1 2 1 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively

  15. Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides

    Science.gov (United States)

    Navarro, Marcos Vicente de A. S.; Vierira, Débora F.; Nagem, Ronaldo A. P.; de Araújo, Ana Paula U.; Oliva, Maria Luiza V.; Garratt, Richard C.

    2005-01-01

    A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V M = 2.5 Å3 Da−1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method. PMID:16511193

  16. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica

    International Nuclear Information System (INIS)

    Bhatt, Harshesh; Trivedi, Dipesh Kumar; Pal, Ravi Kant; Johri, Atul Kumar; Tuteja, Narendra; Bhavesh, Neel Sarovar

    2012-01-01

    Cyclophin A like protein from Piriformospora indica involved in salt-stress tolerance was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 2 Å resolution and belonged to space group C222 1 . Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis–trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V M = 4.48 Å 3 Da −1 , 72.6% solvent content)

  17. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana cyclophilin 38 (AtCyp38)

    International Nuclear Information System (INIS)

    Vasudevan, Dileep; Gopalan, Gayathri; He, Zengyong; Luan, Sheng; Swaminathan, Kunchithapadam

    2005-01-01

    Crystallization of Arabidopsis thaliana cyclophilin 38. The crystal diffracts X-rays to 2.5 Å resolution. AtCyp38 is one of the highly divergent multidomain cyclophilins from Arabidopsis thaliana. A recombinant form of AtCyp38 (residues 83–437) was expressed in Escherichia coli and purified to homogeneity. The protein was crystallized using the vapour-batch technique with PEG 6000 and t-butanol as precipitants. Crystals of recombinant AtCyp38 diffracted X-rays to better than 2.5 Å resolution at 95 K using a synchrotron-radiation source. The crystal belongs to the C-centred orthorhombic space group C222 1 , with unit-cell parameters a = 58.2, b = 95.9, c = 167.5 Å, and contains one molecule in the asymmetric unit. The selenomethionine derivative of the AtCyp38 protein was overexpressed, purified and crystallized in the same space group and data were collected to 3.5 Å at the NSLS synchrotron. The structure is being solved by the MAD method

  18. Crystallization and preliminary X-ray analysis of βC–S lyases from two oral streptococci

    International Nuclear Information System (INIS)

    Kezuka, Yuichiro; Yoshida, Yasuo; Nonaka, Takamasa

    2009-01-01

    The βC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, were cloned, overproduced, purified and crystallized. The obtained crystals were characterized by X-ray diffraction. Hydrogen sulfide, which causes oral malodour, is generally produced from l-cysteine by the action of βC–S lyase from oral bacteria. The βC–S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus βC–S lyase belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 Å, and the crystal of S. gordonii βC–S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 Å. The structures of the βC–S lyases were solved by molecular-replacement techniques

  19. Expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of glyceraldehyde-3-phosphate dehydrogenase from Campylobacter jejuni

    International Nuclear Information System (INIS)

    Tourigny, David S.; Elliott, Paul R.; Edgell, Louise J.; Hudson, Gregg M.; Moody, Peter C. E.

    2010-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray analysis of wild-type and of an active-site mutant of C. jejuni glyceraldehyde-3-phosphate dehydrogenase is reported. The genome of the enteric pathogen Campylobacter jejuni encodes a single glyceraldehyde-3-phosphate dehydrogenase that can utilize either NADP + or NAD + as coenzymes for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of both the wild type and an active-site mutant of the enzyme are presented. Preliminary X-ray analysis revealed that in both cases the crystals diffracted to beyond 1.9 Å resolution. The space group is shown to be I4 1 22, with unit-cell parameters a = 90.75, b = 90.75, c = 225.48 Å, α = 90.46, β = 90.46, γ = 222.79°; each asymmetric unit contains only one subunit of the tetrameric enzyme

  20. Purification, isolation, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 from Drosophila melanogaster

    Science.gov (United States)

    Boyko, K. M.; Nikolaeva, A. Yu.; Kachalova, G. S.; Bonchuk, A. N.; Popov, V. O.

    2017-11-01

    The spatial organization of the genome is controlled by a special class of architectural proteins, including proteins containing BTB domains that are able to dimerize or multimerize. The centrosomal protein 190 is one of such architectural proteins. The purification, crystallization, and preliminary X-ray diffraction study of the BTB domain of the centrosomal protein 190 are reported. The crystallization conditions were found by the vapor-diffusion technique. The crystals diffracted to 1.5 Å resolution and belonged to sp. gr. P3221. The structure was solved by the molecular replacement method. The structure refinement is currently underway.

  1. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Thermotoga neapolitana β-glucosidase B

    Energy Technology Data Exchange (ETDEWEB)

    Turner, Pernilla [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Pramhed, Anna [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Kanders, Erik; Hedström, Martin; Karlsson, Eva Nordberg, E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Logan, Derek T., E-mail: eva.nordberg-karlsson@biotek.lu.se [Department of Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden); Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, S-221 00 Lund (Sweden)

    2007-09-01

    Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. β-Glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C222{sub 1}, with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 Å. Native data have been collected to 2.4 Å resolution and the structure has been solved to 2.7 Å using the selenomethionine MAD method. Model building and refinement of the structure are under way.

  2. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won, E-mail: sewonsuh@snu.ac.kr [Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2006-02-01

    Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B{sub 6} (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V{sub M}) of 3.64 Å{sup 3} Da{sup −1} and a solvent content of 66%.

  3. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-01-01

    Erythronate-4-phosphate dehydrogenase from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.20 Å resolution. The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-α-ketobutyrate. It belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B 6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 Å from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 Å. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (V M ) of 3.64 Å 3 Da −1 and a solvent content of 66%

  4. Crystallization and preliminary X-ray analysis of the stress-response PPM phosphatase RsbX from Bacillus subtilis

    International Nuclear Information System (INIS)

    Suganuma, Masatoshi; Teh, Aik Hong; Makino, Masatomo; Shimizu, Nobutaka; Kaneko, Tomonori; Hirata, Kunio; Yamamoto, Masaki; Kumasaka, Takashi

    2009-01-01

    The bacterial PPM phosphatase RsbX from B. subtilis was expressed in E. coli, purified and crystallized. The crystal belonged to space group P1 and diffracted to 1.06 Å resolution. RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase σ factor SigB. In order to elucidate the structural–functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 Å resolution with an R merge of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 Å, α = 98.8, β = 90.0, γ = 108.4°

  5. Purification, crystallization and preliminary X-ray diffraction study on pyrimidine nucleoside phosphorylase TTHA1771 from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    Shimizu, Katsumi; Kunishima, Naoki

    2007-01-01

    The pyrimidine nucleoside phosphorylase TTHA1771 from T. thermophilus HB8 has been overexpressed, purified and crystallized. The crystals diffract X-rays to 1.8 Å resolution using synchrotron radiation. Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide-synthesis salvage pathway. In order to study the structure–thermostability relationship of this enzyme, PYNP from the extreme thermophile Thermus thermophilus HB8 (TTHA1771) has been cloned, overexpressed and purified. The TTHA1771 protein was crystallized at 291 K using the oil-microbatch method with PEG 4000 as a precipitant. A native data set was collected to 1.8 Å resolution using synchrotron radiation. The crystal belongs to the monoclinic space group P2 1 , with unit-cell parameters a = 58.83, b = 76.23, c = 103.86 Å, β = 91.3°

  6. Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum

    International Nuclear Information System (INIS)

    Sutay Kocabas, Didem; Pearson, Arwen R.; Phillips, Simon E. V.; Bakir, Ufuk; Ogel, Zumrut B.; McPherson, Michael J.; Trinh, Chi H.

    2009-01-01

    The bifunctional enzyme catalase-phenol oxidase from S. thermophilum was crystallized by the hanging-drop vapour-diffusion method in space group P2 1 and diffraction data were collected to 2.8 Å resolution. Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P2 1 and contained one tetramer per asymmetric unit

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum

    International Nuclear Information System (INIS)

    Barata, Lídia; Sousa Silva, Marta; Schuldt, Linda; Costa, Gonçalo da; Tomás, Ana M.; Ferreira, António E. N.; Weiss, Manfred S.; Ponces Freire, Ana; Cordeiro, Carlos

    2010-01-01

    Glyoxalase I from L. infantum was cloned, overexpressed in E. coli, purified and crystallized. Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-d-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized. Two crystal forms were obtained: a cube-shaped form and a rod-shaped form. While the cube-shaped form did not diffract X-rays at all, the rod-shaped form exhibited diffraction to about 2.0 Å resolution. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 130.03, b = 148.51, c = 50.63 Å and three dimers of the enzyme per asymmetric unit

  8. Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

    International Nuclear Information System (INIS)

    Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

    2012-01-01

    The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution. Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit

  9. Escherichia coli tRNAArg acceptor-stem isoacceptors: comparative crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Eichert, André; Schreiber, Angela; Fürste, Jens P.; Perbandt, Markus; Betzel, Christian; Erdmann, Volker A.; Förster, Charlotte

    2009-01-01

    Various E. coli tRNA Arg acceptor-stem microhelix isoacceptors have been crystallized and investigated by high-resolution X-ray diffraction analysis. The aminoacylation of tRNA is a crucial step in cellular protein biosynthesis. Recognition of the cognate tRNA by the correct aminoacyl-tRNA synthetase is ensured by tRNA identity elements. In tRNA Arg , the identity elements consist of the anticodon, parts of the D-loop and the discriminator base. The minor groove of the aminoacyl stem interacts with the arginyl-tRNA synthetase. As a consequence of the redundancy of the genetic code, six tRNA Arg isoacceptors exist. In the present work, three different Escherichia coli tRNA Arg acceptor-stem helices were crystallized. Two of them, the tRNA Arg microhelices RR-1660 and RR-1662, were examined by X-ray diffraction analysis and diffracted to 1.7 and 1.8 Å resolution, respectively. The tRNA Arg RR-1660 helix crystallized in space group P1, with unit-cell parameters a = 26.28, b = 28.92, c = 29.00 Å, α = 105.74, β = 99.01, γ = 97.44°, whereas the tRNA Arg RR-1662 helix crystallized in space group C2, with unit-cell parameters a = 33.18, b = 46.16, c = 26.04 Å, β = 101.50°

  10. Crystallization and preliminary X-ray analysis of the TetR-like efflux pump regulator SimR

    International Nuclear Information System (INIS)

    Le, Tung B. K.; Stevenson, Clare E. M.; Buttner, Mark J.; Lawson, David M.

    2011-01-01

    Crystals of SimR, a TetR-like efflux pump repressor from S. antibioticus, were obtained and X-ray data were recorded to a resolution of 2.3 Å. Crystals of SimR were grown by vapour diffusion. The protein crystallized with trigonal symmetry and X-ray data were recorded to a resolution of 2.3 Å from a single crystal at the synchrotron. SimR belongs to the TetR family of bacterial transcriptional regulators. In the absence of the antibiotic simocyclinone, SimR represses the transcription of a divergently transcribed gene encoding the simocyclinone efflux pump SimX in Streptomyces antibioticus by binding to operators in the simR–simX intergenic region. Simocyclinone binding causes SimR to dissociate from its operators, leading to expression of the SimX efflux pump. Thus, SimR represents an intimate link between the biosynthesis of simocyclinone and its export, which may also provide the mechanism of self-resistance to the antibiotic in the producer strain

  11. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    International Nuclear Information System (INIS)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    The NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra was expressed, purified, and crystallized and X-ray diffraction data of this crystal were collected to 2.2 Å resolution. (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%

  12. Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi; Kita, Akiko [Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); Fukami, Takaaki A.; Mio, Toshiyuki; Yamada-Okabe, Hisafumi [Kamakura Research Laboratory, Chugai Pharmaceutical Co. Ltd, 200 Kajiwara, Kamakura, Kanagawa 247-8530 (Japan); Yamada-Okabe, Toshiko [Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004 (Japan); Miki, Kunio, E-mail: miki@kuchem.kyoto-u.ac.jp [Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502 (Japan); RIKEN SPring-8 Center at Harima Institute, Koto 1-1-1, Sayocho, Sayo-gun, Hyogo 679-5148 (Japan)

    2006-12-01

    UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.

  13. Crystallization and preliminary X-ray diffraction analysis of the haem-binding protein HemS from Yersinia enterocolitica

    International Nuclear Information System (INIS)

    Schneider, Sabine; Paoli, Massimo

    2005-01-01

    The haem binding protein HemS from Y. enterocolitica has been crystallized in complex with its ligand. The crystals diffracted X-rays to 2.6 Å in-house. Bacteria have evolved strategies to acquire iron from their environment. Pathogenic microbes rely on specialized proteins to ‘steal’ haem from their host and use it as an iron source. HemS is the ultimate recipient of a molecular-relay system for haem uptake in Gram-negative species, functioning as the cytosolic carrier of haem. Soluble expression and high-quality diffraction crystals were obtained for HemS from Yersinia enterocolitica. Crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 74.86, b = 77.45, c = 114.09 Å, and diffract X-rays to 2.6 Å spacing in-house. Determination of the structure of the haem–HemS complex will reveal the molecular basis of haem binding

  14. Crystallization and preliminary X-ray diffraction analysis of the haem-binding protein HemS from Yersinia enterocolitica

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Sabine; Paoli, Massimo, E-mail: max.paoli@nottingham.ac.uk [School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)

    2005-08-01

    The haem binding protein HemS from Y. enterocolitica has been crystallized in complex with its ligand. The crystals diffracted X-rays to 2.6 Å in-house. Bacteria have evolved strategies to acquire iron from their environment. Pathogenic microbes rely on specialized proteins to ‘steal’ haem from their host and use it as an iron source. HemS is the ultimate recipient of a molecular-relay system for haem uptake in Gram-negative species, functioning as the cytosolic carrier of haem. Soluble expression and high-quality diffraction crystals were obtained for HemS from Yersinia enterocolitica. Crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 74.86, b = 77.45, c = 114.09 Å, and diffract X-rays to 2.6 Å spacing in-house. Determination of the structure of the haem–HemS complex will reveal the molecular basis of haem binding.

  15. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Carr, Stephen B., E-mail: bmbsbc@bmb.leeds.ac.uk [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Makris, George [Omega Mediation Hellas Ltd, Clinical and Pharma Consulting, 11525 N. Psychiko, Athens (Greece); Phillips, Simon E. V.; Thomas, Christopher D. [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  16. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

    2006-01-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2 1 , diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2 1 2 1 2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism

  17. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    International Nuclear Information System (INIS)

    Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Szyczew, Alex J.; Kiefel, Milton J.; Itzstein, Mark von; Blanchard, Helen

    2005-01-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement

  18. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus) PMB 50, Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S. [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Szyczew, Alex J.; Kiefel, Milton J.; Itzstein, Mark von; Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus) PMB 50, Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-06-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.

  19. Crystallization and preliminary X-ray analysis of the reductase component of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii

    International Nuclear Information System (INIS)

    Oonanant, Worrapoj; Sucharitakul, Jeerus; Chaiyen, Pimchai; Yuvaniyama, Jirundon

    2012-01-01

    The reductase component of p-hydroxyphenylacetate 3-hydroxylase from A. baumannii was overexpressed, purified and crystallized. X-ray diffraction data were collected and processed to 2.3 Å resolution. p-Hydroxyphenylacetate 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) at the ortho position to yield 3,4-dihydroxyphenylacetate (DHPA). HPAH from A. baumannii is a two-component flavoprotein consisting of a smaller reductase (C 1 ) component and a larger oxygenase (C 2 ) component. The C 1 component supplies a reduced flavin in its free form to the C 2 counterpart for hydroxylation. In addition, HPA can bind to C 1 and enhance the flavin-reduction rate without becoming hydroxylated. The recombinant C 1 component was purified and crystallized using the microbatch method at 295 K. X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation on the BL13B1 beamline at NSRRC, Taiwan. The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.78, b = 59.92, c = 211.85 Å, and contained two molecules of C 1 per asymmetric unit

  20. Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2

    International Nuclear Information System (INIS)

    Rodríguez Guilbe, María M.; Alfaro Malavé, Elisa C.; Akerboom, Jasper; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

    2008-01-01

    The genetically encoded fluorescent calcium-indicator protein GCaMP2 was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution and the structure was solved by molecular replacement. Fluorescent proteins and their engineered variants have played an important role in the study of biology. The genetically encoded calcium-indicator protein GCaMP2 comprises a circularly permuted fluorescent protein coupled to the calcium-binding protein calmodulin and a calmodulin target peptide, M13, derived from the intracellular calmodulin target myosin light-chain kinase and has been used to image calcium transients in vivo. To aid rational efforts to engineer improved variants of GCaMP2, this protein was crystallized in the calcium-saturated form. X-ray diffraction data were collected to 2.0 Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 126.1, b = 47.1, c = 68.8 Å, β = 100.5° and one GCaMP2 molecule in the asymmetric unit. The structure was phased by molecular replacement and refinement is currently under way

  1. Crystallization and preliminary X-ray analysis of the MaoC-like dehydratase from Phytophthora capsici

    International Nuclear Information System (INIS)

    Wang, Huizheng; Guo, Jiubiao; Pang, Hai; Zhang, Xiuguo

    2010-01-01

    The MaoC-like dehydratase from P. capsici was cloned, expressed and purified to homogeneity. Crystals were obtained that diffracted to 1.93 Å resolution. MaoC-like dehydratase (MaoC) plays an important role in supplying (R)-3-hydroxyacyl-CoA from the fatty-acid oxidation pathway to polyhydroxyalkanoate (PHA) biosynthetic pathways. PHAs have been attracting much attention as they can be used in the biosynthesis of synthetic plastics. Crystals of MaoC from Phytophora capsici were grown by the hanging-drop vapour-diffusion method at 289 K in a number of screening conditions. An MaoC crystal diffracted to 1.93 Å resolution using X-ray radiation and belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 81.458, b = 82.614, c = 124.228 Å, α = β = γ = 90°

  2. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum

    International Nuclear Information System (INIS)

    Higashi, Yasuhiro; Smith, Thomas J.; Jez, Joseph M.; Kutchan, Toni M.

    2010-01-01

    Recombinant P. somniferum salutaridine reductase (SalR) was purified and crystallized with NADPH using the hanging-drop vapor-diffusion method. Crystals of the SalR–NADPH complex diffracted X-rays to a resolution of 1.9 Å. The opium poppy Papaver somniferum is the source of the narcotic analgesics morphine and codeine. Salutaridine reductase (SalR; EC 1.1.1.248) reduces the C-7 keto group of salutaridine to the C-7 (S)-hydroxyl group of salutaridinol in the biosynthetic pathway that leads to morphine in the opium poppy plant. P. somniferum SalR was overproduced in Escherichia coli and purified using cobalt-affinity and size-exclusion chromatography. Hexagonal crystals belonging to space group P6 4 22 or P6 2 22 were obtained using ammonium sulfate as precipitant and diffracted to a resolution of 1.9 Å

  3. Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida

    International Nuclear Information System (INIS)

    Riise, Ellen Kristin; Lorentzen, Marit Sjo; Helland, Ronny; Willassen, Nils Peder

    2005-01-01

    Monoclinic (P2 1 ) crystals of a His-tagged form of V. salmonicida catalase without cofactor diffract X-rays to 1.96 Å. Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, β = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit

  4. Crystallization and preliminary X-ray diffraction analysis of a cold-adapted catalase from Vibrio salmonicida

    Energy Technology Data Exchange (ETDEWEB)

    Riise, Ellen Kristin [The Norwegian Structural Biology Centre, Faculty of Science, University of Tromsø, N-9037 Tromsø (Norway); Lorentzen, Marit Sjo [Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø (Norway); Helland, Ronny [The Norwegian Structural Biology Centre, Faculty of Science, University of Tromsø, N-9037 Tromsø (Norway); Willassen, Nils Peder, E-mail: nilspw@fagmed.uit.no [The Norwegian Structural Biology Centre, Faculty of Science, University of Tromsø, N-9037 Tromsø (Norway); Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø (Norway)

    2006-01-01

    Monoclinic (P2{sub 1}) crystals of a His-tagged form of V. salmonicida catalase without cofactor diffract X-rays to 1.96 Å. Catalase (EC 1.11.1.6) catalyses the breakdown of hydrogen peroxide to water and molecular oxygen. Recombinant Vibrio salmonicida catalase (VSC) possesses typical cold-adapted features, with higher catalytic efficiency, lower thermal stability and a lower temperature optimum than its mesophilic counterpart from Proteus mirabilis. Crystals of VSC were produced by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 98.15, b = 217.76, c = 99.28 Å, β = 110.48°. Data were collected to 1.96 Å and a molecular-replacement solution was found with eight molecules in the asymmetric unit.

  5. Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein

    International Nuclear Information System (INIS)

    Abd Aziz, Abd Ghani; Ruzheinikov, Sergey N.; Sedelnikova, Svetlana E.; Mohamed, Rahmah; Nathan, Sheila; Baker, Patrick J.; Rice, David W.

    2012-01-01

    The L1 ribosomal protein from B. pseudomallei has been overexpressed, purified and crystallized in a form suitable for X-ray analysis. The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit

  6. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD

    International Nuclear Information System (INIS)

    Ruiz-Ramos, Alba; Lallous, Nada; Grande-García, Araceli; Ramón-Maiques, Santiago

    2013-01-01

    The recombinant aspartate transcarbamoylase domain of human CAD was expressed in E. coli, purified and crystallized in the presence and absence of the inhibitor PALA. X-ray diffraction data sets were collected for both crystal forms at 2.1 Å resolution. Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-l-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6 3 22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P2 1 lattice with pseudo-symmetry and possibly twinning

  7. Purification, crystallization and preliminary X-ray crystallographic analysis of the ATPase domain of human TAP in nucleotide-free and ADP-, vanadate- and azide-complexed forms

    International Nuclear Information System (INIS)

    Meena, Sita R.; Gangwar, Shanti P.; Saxena, Ajay K.

    2012-01-01

    The ATPase domain of human TAP in nucleotide free, ADP, vanadate and azide complexed forms were purified and crystallized. The X-ray diffraction data sets were collected for all crystals in the resolution range of 2.8–3.0 Å. The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP–Mg 2+ , mimicking the product-bound state; (iii) in complex with vanadate–ADP–Mg 2+ , mimicking the ATP-bound state; and (iv) in complex with azide–ADP–Mg 2+ , also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 Å. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6 2 , with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported

  8. Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

    International Nuclear Information System (INIS)

    Marques-Carvalho, Maria João; Morais-Cabral, João Henrique

    2012-01-01

    The crystallization conditions and preliminary crystal characterization of the cytoplasmic cyclic nucleotide-binding homology domain from the mouse EAG potassium channel are reported. The members of the family of voltage-gated KCNH potassium channels play important roles in cardiac and neuronal repolarization, tumour proliferation and hormone secretion. These channels have a C-terminal cytoplasmic domain which is homologous to cyclic nucleotide-binding domains (CNB-homology domains), but it has been demonstrated that channel function is not affected by cyclic nucleotides and that the domain does not bind nucleotides in vitro. Here, the crystallization and preliminary crystallographic analysis of a CNB-homology domain from a member of the KCNH family, the mouse EAG channel, is reported. X-ray diffraction data were collected to 2.2 Å resolution and the crystal belonged to the hexagonal space group P3 1 21

  9. Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus

    International Nuclear Information System (INIS)

    Jackson, Michael R.; Selby, Thomas L.

    2012-01-01

    Crystallization and diffraction analysis of a Ca 2+ -dependent PI-PLC from Streptomyces is reported. Optimization of crystals was completed using a drop-pinning technique and heavy-atom soaks to achieve high-quality diffraction to 1.23 Å. A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) from Streptomyces antibioticus has been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 41.26, b = 51.86, c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution

  10. Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)

    International Nuclear Information System (INIS)

    Couñago, Rafael Miguez; Fleming, Stephen B.; Mercer, Andrew A.; Krause, Kurt L.

    2010-01-01

    The chemokine-binding protein from orf virus was purified and crystallized. The morphology and diffraction behaviour of these crystals was significantly improved through the use of additives known as Silver Bullets. The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the host’s immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP–chemokine binding, ORFV CBP was crystallized as part of an ongoing structure–function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50 Å resolution and belonged to the hexagonal space group P6 1 22 or its enantiomorph P6 5 22, with unit-cell parameters a = b = 75.62, c = 282.49 Å, α = 90, β = 90, γ = 120°

  11. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    Science.gov (United States)

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

  12. Crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltE from Escherichia coli

    International Nuclear Information System (INIS)

    Artola-Recolons, Cecilia; Llarrull, Leticia I.; Lastochkin, Elena; Mobashery, Shahriar; Hermoso, Juan A.

    2010-01-01

    Crystals of the lytic transglycosylase MltE from E. coli were grown using the microbatch method and diffracted to a resolution of 2.1 Å. MltE from Escherichia coli (193 amino acids, 21 380 Da) is a lytic transglycosylase that initiates the first step of cell-wall recycling. This enzyme is responsible for the cleavage of the cell-wall peptidoglycan at the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units. At the end this reaction generates a disaccharide that is internalized and initiates the recycling process. To obtain insights into the biological functions of MltE, crystallization trials were performed and crystals of MltE protein that were suitable for X-ray diffraction analysis were obtained. The MltE protein of E. coli was crystallized using the hanging-drop vapour-diffusion method at 291 K. Crystals grew from a mixture consisting of 28% polyethylene glycol 4000, 0.1 M Tris pH 8.4 and 0.2 M magnesium chloride. Further optimization was performed using the microbatch technique. Single crystals were obtained that belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 123.32, b = 183.93, c = 35.29 Å, and diffracted to a resolution of 2.1 Å

  13. Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

    International Nuclear Information System (INIS)

    Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing; Qiu, Bingsheng; Rao, Zihe; Gao, George F.; Tien, Po

    2005-01-01

    Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion

  14. Crystallization and preliminary X-ray crystallographic analysis of Aquifex aeolicus SelA, a bacterial selenocysteine synthase

    International Nuclear Information System (INIS)

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2012-01-01

    The bacterial selenocysteine synthase SelA from Aquifex aeolicus was crystallized and the diffraction resolution was improved by lysine-residue methylation, truncation of N-terminal region (ΔN), and Lys-to-Ala point mutations. Phases were determined by using a selenomethionine-substituted crystal of the ΔN mutant. Selenocysteine (Sec), the 21st amino acid, is synthesized on its specific tRNA (tRNA Sec ) via a multi-step process. In bacteria, tRNA Sec is ligated first with serine by seryl-tRNA synthetase, which is followed by Ser-to-Sec conversion by Sec synthase (SelA). To elucidate its structure and catalytic mechanism, Aquifex aeolicus SelA was crystallized. Although wild-type SelA crystals diffracted X-rays poorly (to up to 8 Å resolution), the resolution was improved by introducing a quadruple point mutation targeting the loop regions and by methylating the lysine residues, which yielded 3.9 Å resolution diffraction data from a full-length SelA crystal. Truncation of the N-terminal region (ΔN) also improved the resolution. A 3.3 Å resolution data set for phase determination was obtained from a crystal of selenomethionine-substituted Lys-methylated SelA-ΔN

  15. X-ray streak crystal spectography

    International Nuclear Information System (INIS)

    Kauffman, R.L.; Brown, T.; Medecki, H.

    1983-01-01

    We have built an x-ray streaked crystal spectrograph for making time-resolved x-ray spectral measurements. This instrument can access Bragg angles from 11 0 to 38 0 and x-ray spectra from 200 eV to greater than 10 keV. We have demonstrated resolving powers, E/δE > 200 at 1 keV and time resolution less than 20 psec. A description of the instrument and an example of the data is given

  16. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    International Nuclear Information System (INIS)

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter; Chow, Joseph W.; Lerner, Stephen; Vakulenko, Sergei; Smith, Clyde A.

    2005-01-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2 1 , with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding

  17. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    Energy Technology Data Exchange (ETDEWEB)

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter [Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand); Chow, Joseph W.; Lerner, Stephen [Division of Infectious Diseases, Wayne State University School of Medicine and VA Medical Center, Detroit, Michigan 48201 (United States); Vakulenko, Sergei [Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 (United States); Smith, Clyde A., E-mail: csmith@slac.stanford.edu [Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025 (United States); Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland (New Zealand)

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  18. Crystallization and preliminary X-ray studies of a galactose-specific lectin from the seeds of bitter gourd (Momordica charantia)

    International Nuclear Information System (INIS)

    Chandran, Thyageshwar; Sharma, Alok; Vijayan, M.

    2010-01-01

    A galactose-specific lectin purified from the seeds of bitter gourd (M. charantia) has been crystallized and preliminary X-ray study of the crystals has been carried out. A galactose-specific lectin from the seeds of bitter gourd (Momordica charantia) is a four-chain type II ribosome-inactivating protein (RIP) resulting from covalent association through a disulfide bridge between two identical copies of a two-chain unit. The available structural information on such four-chain RIPs is meagre. The bitter gourd lectin was therefore crystallized for structural investigation and the crystals have been characterized. It is anticipated that the structure of the orthorhombic crystals will be analysed using molecular replacement by taking advantage of its sequence, and presumably structural, homology to normal two-chain type II RIPs

  19. Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio

    International Nuclear Information System (INIS)

    Okada, Akitoshi; Nagata, Koji; Sano, Kaori; Yasumasu, Shigeki; Kubota, Keiko; Ohtsuka, Jun; Iuchi, Ichiro; Tanokura, Masaru

    2009-01-01

    The hatching enzyme of zebrafish, ZHE1, was expressed, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belonged to space group P2 1 2 1 2 1 and diffracted X-rays to a resolution of 1.14 Å. The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0–1.80 and 50.0–1.14 Å were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0–1.14 Å. The space group was assigned as primitive orthorhombic P2 1 2 1 2 1 , with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 Å. The crystal contained one ZHE1 molecule in the asymmetric unit

  20. Crystallization and preliminary X-ray analysis of a novel esterase Rv0045c from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Xu, Lipeng; Guo, Jiubiao; Zheng, Xiangdong; Wen, Tingyi; Sun, Fei; Liu, Siguo; Pang, Hai

    2010-01-01

    The novel esterase Rv0045c from M. tuberculosis was expressed and purified to homogeneity. The crystals of native and SeMet-labelled Rv0045c protein that were obtained diffracted to resolutions of 2.7 and 3.0 Å, respectively. The Rv0045c protein is predicted to be an esterase that is involved in lipid metabolism in Mycobacterium tuberculosis. The protein was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The Rv0045c protein crystals diffracted to a resolution of 2.7 Å using a synchrotron-radiation source and belonged to space group P3 1 or P3 2 , with unit-cell parameters a = b = 73.465, c = 48.064 Å, α = β = 90, γ = 120°. Purified SeMet-labelled Rv0045c protein was also crystallized and formed crystals that diffracted to a resolution of 3.0 Å using an in-house X-ray radiation source

  1. Crystallization and preliminary X-ray crystallographic studies of β-transaminase from Mesorhizobium sp. strain LUK

    International Nuclear Information System (INIS)

    Kim, Bokyung; Park, Ok Kyeung; Bae, Ju Young; Jang, Tae-ho; Yoon, Jong Hwan; Do, Kyoung Hun; Kim, Byung-Gee; Yun, Hyungdon; Park, Hyun Ho

    2011-01-01

    β-Transaminase from Mesorhizobium sp. strain LUK was crystallized. The crystals were found to belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å. The crystals were obtained at 293 K and diffracted to a resolution of 2.5 Å. β-Transaminase (β-TA) catalyzes the transamination reaction between β-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure β-amino acids for pharmaceutical purposes. The β-TA from Mesorhizobium sp. strain LUK (β-TAMs) belongs to a novel class in that it shows β-transaminase activity with a broad and unique substrate specificity. In this study, β-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. β-TAMs was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.5 Å from a crystal that belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å

  2. Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum

    International Nuclear Information System (INIS)

    Yeo, Hyun Ku; Kang, Jina; Park, Young Woo; Sung, Jung-Suk; Lee, Jae Young

    2012-01-01

    Orthorhombic crystals of DtxR from T. acidophilum have been obtained. X-ray data were collected to 1.8 Å resolution using synchrotron radiation. The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = β = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (V M ) of 2.22 Å 3 Da −1 and a solvent content of 44.6%

  3. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    Energy Technology Data Exchange (ETDEWEB)

    Ku, Min-Je [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Won-Ho [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Nam, Ki-hyun; Rhee, Kyeong-hee [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Ki-Seog [Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Eunice EunKyung [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Yu, Myung-Hee [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Hwang, Kwang Yeon, E-mail: hwangky@kist.re.kr [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of)

    2005-04-01

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn{sup 2+} ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (V{sub M}) of 3.3 Å{sup 3} Da{sup −1} and a solvent content of 62.7%.

  4. Crystallization and preliminary X-ray crystallographic studies of VibE, a vibriobactin-specific 2,3-dihydroxybenzoate-AMP ligase from Vibrio cholerae

    International Nuclear Information System (INIS)

    Liu, Xiuhua; Wang, Zhi; Zhu, Deyu; Wei, Tiandi; Gu, Lichuan; Xu, Sujuan

    2011-01-01

    This article reports the molecular cloning, protein expression and purification, crystallization and preliminary X-ray crystallographic analysis of the vibriobactin synthetase VibE from V. cholerae. Vibriobactin synthetases (VibABCDEFH) catalyze the biosynthesis of vibriobactin in the pathogenic bacterium Vibrio cholerae. VibE, a vibriobactin-specific 2,3-dihydroxybenzoate-AMP ligase, plays a critical role in the transfer of 2,3-dihydroxybenzoate to the aryl carrier protein domain of holo VibB. Here, the cloning, protein expression and purification, crystallization and preliminary X-ray crystallographic analysis of VibE from V. cholerae are reported. The VibE crystal diffracted to 2.3 Å resolution. The crystal belonged to space group P2 1 , with unit-cell parameters a = 56.471, b = 45.927, c = 77.014 Å, β = 95.895°. There is one protein molecule in the asymmetric unit, with a corresponding Matthews coefficient of 1.63 Å 3 Da −1 and solvent content of 24.41%

  5. Cloning, purification, crystallization and preliminary X-ray studies of flagellar hook scaffolding protein FlgD from Pseudomonas aeruginosa PAO1

    International Nuclear Information System (INIS)

    Luo, Miao; Niu, Siqiang; Yin, Yibing; Huang, Ailong; Wang, Deqiang

    2009-01-01

    In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. FlgD regulates the assembly of the hook cap structure to prevent leakage of hook monomers into the medium and hook monomer polymerization and also plays a role in determination of the correct hook length, with the help of the FliK protein. In order to better elucidate the functions of FlgD in flagellar hook biosynthesis, the three-dimensional structure of FlgD is being determined by X-ray crystallography. Here, the expression, purification, crystallization and preliminary crystallographic analysis of FlgD from P. aeruginosa are reported. The crystal belonged to space group I222 and diffracted to a resolution of 2.5 Å, with unit-cell parameters a = 116.47, b = 118.71, c = 118.85 Å. The crystals are most likely to contain three molecules in the asymmetric unit, with a V M value of 2.73 Å 3 Da −1

  6. Crystallization and preliminary X-ray crystallographic analysis of a highly specific serpin from the beetle Tenebrio molitor

    Science.gov (United States)

    Park, Sun Hee; Piao, Shunfu; Kwon, Hyun-Mi; Kim, Eun-Hye; Lee, Bok Luel; Ha, Nam-Chul

    2010-01-01

    The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spätzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpho­lino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 Å resolution and were suitable for structure determination. The crystals belonged to space group P21. The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease. PMID:20124722

  7. Crystallization and preliminary X-ray analysis of the vWA domain of human anthrax toxin receptor 1

    International Nuclear Information System (INIS)

    Cai, Chenguang; Zhao, Ying; Tong, Xiaohang; Fu, Sheng; Li, Yuanyuan; Wu, Yang; Li, Xumei; Lou, Zhiyong

    2010-01-01

    The vWA domain of human anthrax toxin receptor 1 was overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 1.8 Å resolution. The Gram-positive spore-forming bacterium Bacillus anthracis causes anthrax by secreting anthrax toxin, which consists of protective antigen (PA), lethal factor and oedema factor. Binding of PA to receptors triggers the multi-step process of anthrax toxin entry into target cells. Two distinct cellular receptors, ANTXR1 (also known as tumour endothelial marker 8; TEM8) and ANTXR2 (also known as capillary morphogenesis protein 2; CMG2), for anthrax toxin have been identified. Although the crystal structure of the extracellular von Willebrand factor A (vWA) domain of CMG2 has been reported, the difference between the vWA domains of TEM8 and CMG2 remains unclear because there are no structural data for the TEM8 vWA domain. In this report, the TEM8 vWA domain was expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution from a single crystal, which belonged to space group P1 with unit-cell parameters a = 65.9, b = 66.1, c = 74.4 Å, α = 63.7, β = 88.2, γ = 59.9°

  8. Crystallization and preliminary X-ray crystallographic analysis of SMU.412c protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Ye, Zhao-Yang; Hou, Qiao-Ming; Li, Lan-Fen; Su, Xiao-Dong

    2009-01-01

    Crystallization of SMU.412c protein from the caries pathogen Streptococcus mutans can easily appear in the condition 2.8 M sodium acetate pH 7.0 and its crystal belongs to space group P4 1 2 1 2. The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His 6 tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8 Å resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 53.5, c = 141.1 Å

  9. Purification, crystallization and preliminary X-ray analysis of the β-lactamase Oih-1 from Oceanobacillus iheyensis

    International Nuclear Information System (INIS)

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2009-01-01

    Oih-1, a β-lactamase enzyme isolated from the deep-sea bacterium O. iheyensis, has been crystallized and a complete X-ray diffraction data set has been collected to 1.65 Å resolution. Bacterial resistance to the β-lactam family of antibiotics is primarily the result of the deactivation of the drugs by β-lactamase enzymes. The gene encoding the proficient β-lactamase Oih-1 from the alkaliphilic and halotolerant Gram-positive bacterium Oceanobacillus iheyensis has been cloned and the mature wild-type protein (comprising 274 amino-acid residues) has been expressed in Escherichia coli and subsequently purified to homogeneity. Oih-1 crystallized in two crystal forms both belonging to the trigonal space group P3 1 21 but with distinctly different unit-cell parameters. Synchrotron diffraction data were collected to high resolution (1.65–1.75 Å) from both crystal forms on beamlines BL7-1 and BL11-1 at SSRL (Stanford, California, USA)

  10. Overexpression, crystallization and preliminary X-ray crystallographic analysis of erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Ha, Jun Yong; Lee, Ji Hyun; Kim, Kyoung Hoon; Kim, Do Jin; Lee, Hyung Ho; Kim, Hye-Kyung; Yoon, Hye-Jin; Suh, Se Won

    2006-02-01

    The enzyme erythronate-4-phosphate dehydrogenase catalyses the conversion of erythronate-4-phosphate to 3-hydroxy-4-phospho-hydroxy-alpha-ketobutyrate. It belongs to the D-isomer-specific 2-hydroxyacid dehydrogenase family. It is essential for de novo biosynthesis of vitamin B6 (pyridoxine). Erythronate-4-phosphate dehydrogenase from Pseudomonas aeruginosa, a homodimeric enzyme consisting of two identical 380-residue subunits, has been overexpressed in Escherichia coli with a C-terminal purification tag and crystallized at 297 K using 0.7 M ammonium dihydrogen phosphate, 0.4 M ammonium tartrate, 0.1 M sodium citrate pH 5.6 and 10 mM cupric chloride. X-ray diffraction data were collected to 2.20 A from a crystal grown in the presence of NADH. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 84.77, b = 101.28, c = 142.58 A. A dimeric molecule is present in the asymmetric unit, giving a crystal volume per protein weight (VM) of 3.64 A3 Da(-1) and a solvent content of 66%.

  11. Purification, crystallization and preliminary X-ray analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase of Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Zhang, Liping; Feng, Lingling; Zhou, Li; Gui, Jie; Wan, Jian; Hu, Xiaopeng

    2010-01-01

    3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of Streptococcus pneumoniae has been cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. Class II 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases are potential targets for novel antibiotic development. In order to obtain a precise structural model for use in virtual screening and inhibitor design, HMG-CoA reductase of Streptococcus pneumoniae was cloned, overexpressed and purified to homogeneity using Ni–NTA affinity chromatography. Crystals were obtained using the hanging-drop vapour-diffusion method. A complete data set was collected from a single frozen crystal on a home X-ray source. The crystal diffracted to 2.3 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 773.4836, b = 90.3055, c = 160.5592 Å, α = β = γ = 90°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be 54.1% (V M = 2.68 Å 3 Da −1 )

  12. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes

    International Nuclear Information System (INIS)

    Nojiri, Masaki; Shirota, Felicia; Hira, Daisuke; Suzuki, Shinnichiro

    2009-01-01

    The soluble domain of a putative copper-containing nitrite reductase from P. acnes has been overexpressed, purified and crystallized. The crystal belonged to space group P2 1 3 and diffracted to 2.4 Å resolution. The soluble domain (residues 483–913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 Å. The crystal belonged to space group P2 1 3, with unit-cell parameters a = b = c = 108.63 Å. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V M of 2.1 Å 3 Da −1

  13. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN (United Kingdom)

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2{sub 1}; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3{sub 2}. The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP.

  14. Expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    International Nuclear Information System (INIS)

    Elliott, Paul R.; Evans, Daniel; Greenwood, Jacqueline A.; Moody, Peter C. E.

    2008-01-01

    Glyceraldehyde-3-phosphate dehydrogenase A has been cloned, expressed and purified. Apoprotein crystals have been grown which diffracted to 1.75 Å resolution and belonged to space group P2 1 ; holo crystals were grown in the presence of NADP, diffracted to 2.6 Å resolution and belonged to space group P3 2 . The classical glycolytic pathway contains an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, with NADP-dependent forms reserved for photosynthetic organisms and archaea. Here, the cloning, expression, purification, crystallization and preliminary X-ray analysis of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori is reported; crystals of the protein were grown both in the presence and the absence of NADP

  15. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Polekhina, Galina, E-mail: gpolekhina@svi.edu.au; Feil, Susanne C.; Gupta, Abhilasha [St Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065 (Australia); O’Donnell, Paul [Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville 3010 (Australia); Stapleton, David; Parker, Michael W. [St Vincent’s Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065 (Australia)

    2005-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein.

  16. Crystallization of the glycogen-binding domain of the AMP-activated protein kinase β subunit and preliminary X-ray analysis

    International Nuclear Information System (INIS)

    Polekhina, Galina; Feil, Susanne C.; Gupta, Abhilasha; O’Donnell, Paul; Stapleton, David; Parker, Michael W.

    2004-01-01

    The glycogen-binding domain of the AMP-activated kinase β subunit has been crystallized in the presence of β-cyclodextrin. The structure has been determined by single isomorphous replacement and threefold averaging using in-house X-ray data collected from selenomethionine-substituted protein. AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolism in response to energy demand and supply by adjusting the ATP-generating and ATP-consuming pathways. AMPK potentially plays a critical role in diabetes and obesity as it is known to be activated by metforin and rosiglitazone, drugs used for the treatment of type II diabetes. AMPK is a heterotrimer composed of a catalytic α subunit and two regulatory subunits, β and γ. Mutations in the γ subunit are known to cause glycogen accumulation, leading to cardiac arrhythmias. Recently, a functional glycogen-binding domain (GBD) has been identified in the β subunit. Here, the crystallization of GBD in the presence of β-cyclodextrin is reported together with preliminary X-ray data analysis allowing the determination of the structure by single isomorphous replacement and threefold averaging using in-house X-ray data collected from a selenomethionine-substituted protein

  17. Expression, purification, crystallization and preliminary X-ray characterization of the GRP carbohydrate-recognition domain from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Dongwen; Sun, Jianping; Zhao, Wei; Zhang, Xiao; Shi, Yunyu; Teng, Maikun, E-mail: mkteng@ustc.edu.cn; Niu, Liwen, E-mail: mkteng@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027 (China); Dong, Yuhui; Liu, Peng [Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, 19B Yuquan Road, Beijing 100039 (China); Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230027 (China)

    2006-05-01

    The CRD domain of GRP from H. sapiens has been expressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.0 Å. Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for β-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38–172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris–HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 Å at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 Å, β = 118.72°. The estimated Matthews coefficient was 2.6 Å{sup 3} Da{sup −1}, corresponding to 51.8% solvent content.

  18. Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

    International Nuclear Information System (INIS)

    Fernandes, Carlos A. H.; Gartuzo, Elaine C. G.; Pagotto, Ivan; Comparetti, Edson J.; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Costa, Tássia R.; Marangoni, Sergio; Soares, Andreimar M.; Fontes, Marcos R. M.

    2012-01-01

    Two myotoxic and noncatalytic Lys49-phospholipases A 2 (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A 2 (braziliantoxin-III) from B. brazili were crystallized. X-ray diffraction data sets were collected and molecular-replacement solutions were obtained. Two myotoxic and noncatalytic Lys49-phospholipases A 2 (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A 2 (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56–2.05 Å and belonged to space groups P3 1 21 (braziliantoxin-II), P6 5 22 (braziliantoxin-III) and P2 1 (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A 2 (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A 2 braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A 2

  19. Crystallization and preliminary X-ray crystallographic studies of transportin 1 in complex with nucleocytoplasmic shuttling and nuclear localization fragments

    International Nuclear Information System (INIS)

    Imasaki, Tsuyoshi; Shimizu, Toshiyuki; Hashimoto, Hiroshi; Hidaka, Yuji; Yamada, Michiyuki; Sato, Mamoru

    2006-01-01

    Transportin 1 was cocrystallized with nucleocytoplasmic shuttling fragments of JKTBP and hnRNP D and a nuclear localization fragment of TAP. X-ray diffraction data were collected using synchrotron radiation at SPring-8. Nucleocytoplasmic transport of proteins with molar masses of larger than 60 000 is mediated by transport receptors. The transport receptor transportin1 (Trn1) transports various kinds of RNA-binding proteins such as JKTBP, hnRNP D and TAP. Trn1 was successfully cocrystallized with nucleocytoplasmic shuttling fragments of JKTBP and hnRNP D and a nuclear localization fragment of TAP. The crystal of the Trn1–JKTBP fragment complex belongs to space group P2 1 2 1 2, with unit-cell parameters a = 131.5, b =171.5, c = 68.2 Å. The crystals of Trn1 in complex with hnRNP D and TAP fragments are orthorhombic, space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.1, b = 119.1, c = 151.1 Å and a = 69.0, b = 119.1, c = 146.0 Å, respectively. The crystals diffracted to beyond 3.0, 3.2 and 2.4 Å resolution, respectively, using synchrotron radiation at SPring-8

  20. Expression, purification, crystallization and preliminary X-ray characterization of the GRP carbohydrate-recognition domain from Homo sapiens

    International Nuclear Information System (INIS)

    Zhou, Dongwen; Sun, Jianping; Zhao, Wei; Zhang, Xiao; Shi, Yunyu; Teng, Maikun; Niu, Liwen; Dong, Yuhui; Liu, Peng

    2006-01-01

    The CRD domain of GRP from H. sapiens has been expressed, purified and crystallized and X-ray diffraction data have been collected to a resolution of 2.0 Å. Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for β-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38–172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris–HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 Å at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 Å, β = 118.72°. The estimated Matthews coefficient was 2.6 Å 3 Da −1 , corresponding to 51.8% solvent content

  1. Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteria

    International Nuclear Information System (INIS)

    Li, Gui-Lan; Wang, Juan; Li, Lan-Fen; Su, Xiao-Dong

    2009-01-01

    All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. Two dUTPase-encoding genes, yncF and yosS, have been identified in Bacillus subtilis. The gene dut, encoding dUTPase from the dental pathogen Streptococcus mutans, was amplified from S. mutans genomic DNA. The three genes were cloned into expression vectors and overexpressed at high levels in Escherichia coli. Each protein was purified in two steps using chromatographic methods. Crystals of the YosS and YncF proteins and of S. mutans dUTPase were obtained using the vapour-diffusion method. X-ray diffraction data sets were collected from crystals of selenomethionine-labelled YosS and S. mutans dUTPase to resolutions of 2.3 and 1.7 Å, respectively. The crystal of native YncF diffracted to 2.7 Å resolution

  2. Crystallization and preliminary X-ray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coli

    International Nuclear Information System (INIS)

    You, Weijie; Qiu, Xiaoting; Zhang, Yujie; Ma, Jinming; Gao, Yongxiang; Zhang, Xiao; Niu, Liwen; Teng, Maikun

    2010-01-01

    The putative aldose 1-epimerase YeaD from Escherichia coli was crystallized and diffraction data were collected to a resolution of 1.9 Å. Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts α-galactose to β-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected to a resolution of 1.9 Å from a single flash-cooled crystal that belonged to space group P2 1 2 1 2 1

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the Kelch-like motif region of mouse Keap1

    Energy Technology Data Exchange (ETDEWEB)

    Padmanabhan, Balasundaram; Scharlock, Maria [RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Tong, Kit I. [Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance and ERATO Environmental Research Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577 (Japan); Nakamura, Yoshihiro [RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Kang, Moon-Il; Kobayashi, Akira [Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance and ERATO Environmental Research Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577 (Japan); Matsumoto, Takehisa; Tanaka, Akiko [RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Yamamoto, Masayuki [Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance and ERATO Environmental Research Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577 (Japan); Yokoyama, Shigeyuki, E-mail: yokoyama@biochem.s.u-tokyo.ac.jp [RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Cellular Signaling Laboratory and Structurome Research Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2005-01-01

    Keap1-DC (Kelch/double-glycine repeat and C-terminal region) of mouse Keap1 has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method. Keap1 (Kelch-like ECH-associating protein 1) is a negative regulator of the Nrf2 transcription factor in the cytoplasm. The Kelch/DGR (double-glycine repeat) domain of Keap1 associates with Nrf2 as well as with actin filaments. A recombinant protein containing both the Kelch/DGR domain and the C-terminal region of mouse Keap1 was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystal belongs to space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 102.95, c = 55.21 Å, and contains one molecule in the asymmetric unit. A complete diffraction data was collected to 2.25 Å resolution using an R-AXIS IV{sup ++} imaging plate mounted on an RA-Micro7 Cu Kα rotating-anode X-ray generator.

  4. Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wenxin; Wang, Qihai; Bi, Ruchang, E-mail: rcbi@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2005-12-01

    The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap{sub 4}A) hydrolase (EC 3.6.1.41) hydrolyzes Ap{sub 4}A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap{sub 4}A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap{sub 4}A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2{sub 1}, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°.

  5. Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476

    International Nuclear Information System (INIS)

    Bhattacharyya, Sudipta; Dutta, Debajyoti; Ghosh, Ananta Kumar; Das, Amit Kumar

    2011-01-01

    The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from S. aureus MSSA476 is reported. The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li 2 SO 4 , 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV ++ detector. The diffraction data were consistent with the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = β = γ = 90°, and the crystal contained two molecules in the asymmetric unit

  6. Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactose

    International Nuclear Information System (INIS)

    Krejčiříková, Veronika; Fábry, Milan; Marková, Vladimíra; Malý, Petr; Řezáčová, Pavlína; Brynda, Jiří

    2008-01-01

    Mouse galectin-4 carbohydrate binding domain was overexpressed in E. coli and crystallized in the presence of lactose. The crystals belong to tetragonal space group P42 1 2 and diffraction data were collected to 2.1 Å resolution. Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42 1 2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 Å and preliminary X-ray diffraction data were collected to 3.2 Å resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 Å. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of universal stress protein F (YnaF) from Salmonella typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Sagurthi, Someswar Rao; Panigrahi, Rashmi Rekha; Gowda, Giri [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India); Savithri, H. S. [Department of Biochemistry, Indian Institute of Science, Bangalore 560012 (India); Murthy, M. R. N., E-mail: mrn@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012 (India)

    2007-11-01

    The cloning, purification and crystallization of YnaF from S. typhimurium are reported along with preliminary X-ray crystallographic studies. The universal stress protein UspF (YnaF) is a small cytoplasmic bacterial protein. The expression of stress proteins is enhanced when cells are exposed to heat shock, nutrition starvation and certain other stress-inducing agents. YnaF promotes cell survival during prolonged exposure to stress and may activate a general mechanism for stress endurance. This manuscript reports preliminary crystallographic studies on YnaF from Salmonella typhimurium. The gene coding for YnaF was cloned and overexpressed and the protein was purified by Ni–NTA affinity chromatography. Purified YnaF was crystallized using vapour-diffusion and microbatch methods. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.51, b = 77.18, c = 56.34 Å, β = 101.8°. A data set was collected to 2.5 Å resolution with 94.6% completeness using an image-plate detector system mounted on a rotating-anode X-ray generator. Attempts to determine the structure are in progress.

  8. Preparation, crystallization and preliminary X-ray diffraction studies of the glycosylated form of human interleukin-23

    International Nuclear Information System (INIS)

    Shirouzono, Takumi; Chirifu, Mami; Nakamura, Chiharu; Yamagata, Yuriko; Ikemizu, Shinji

    2012-01-01

    Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. IL-23 plays crucial roles in the activation, proliferation and survival of IL-17-producing helper T cells which induce various autoimmune diseases. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells, which produce high-mannose-type glycosylated proteins in order to diminish the heterogeneity of modified N-linked glycans. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to 2.6 Å resolution. The crystal belonged to space group P6 1 or P6 5 , with unit-cell parameters a = b = 108.94, c = 83.79 Å, γ = 120°. Assuming that the crystal contains one molecule per asymmetric unit, the calculated Matthews coefficient was 2.69 Å 3 Da −1 , with a solvent content of 54.2%. The structure was determined by the molecular-replacement method, with an initial R factor of 52.6%. After subsequent rigid-body and positional refinement, the R work and R free values decreased to 31.4% and 38.7%, respectively

  9. Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei

    Energy Technology Data Exchange (ETDEWEB)

    Knight, M. J.; Ruaux, A.; Mikolajek, H.; Erskine, P. T.; Gill, R.; Wood, S. P. [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom); Wood, M. [Institute of Animal Health, Division of Environmental Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN (United Kingdom); Cooper, J. B., E-mail: j.b.cooper@soton.ac.uk [School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX (United Kingdom)

    2006-08-01

    BipD is likely to be a component of a type-III protein secretion system (TTSS) in B. pseudomallei. Native and selenomethionyl-BipD proteins have been expressed and crystals have been obtained which diffract to 2.1 Å. Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 Å resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 Å.

  10. Crystallization and preliminary X-ray diffraction analysis of BipD, a virulence factor from Burkholderia pseudomallei

    International Nuclear Information System (INIS)

    Knight, M. J.; Ruaux, A.; Mikolajek, H.; Erskine, P. T.; Gill, R.; Wood, S. P.; Wood, M.; Cooper, J. B.

    2006-01-01

    BipD is likely to be a component of a type-III protein secretion system (TTSS) in B. pseudomallei. Native and selenomethionyl-BipD proteins have been expressed and crystals have been obtained which diffract to 2.1 Å. Burkholderia pseudomallei, the causative agent of melioidosis, possesses a protein-secretion apparatus that is similar to those found in Salmonella and Shigella. A major function of these secretion systems is to secrete virulence-associated proteins into target cells of the host organism. The BipD gene of B. pseudomallei encodes a secreted virulence factor that is similar in sequence and most likely functionally analogous to IpaD from Shigella and SipD from Salmonella. Thus, the BipD protein is likely to be a component of a type III protein-secretion system (TTSS) in B. pseudomallei. Proteins in the same class as BipD, such as IpaD and SipD, are thought to act as extracellular chaperones to help the hydrophobic translocator proteins enter the target cell membrane, where they form a pore and might even link the translocon pore with the secretion needle. There is evidence that the translocator proteins also bind an integrin which stimulates actin-mediated insertion of the bacterium into the host-cell membrane. Native BipD has been crystallized in a monoclinic crystal form that diffracts X-rays to 2.5 Å resolution. BipD protein which incorporates selenomethionine (SeMet-BipD) has also been expressed and forms crystals which diffract to a higher resolution of 2.1 Å

  11. Isolation, crystallization and preliminary X-ray diffraction analysis of l-amino-acid oxidase from Vipera ammodytes ammodytes venom

    International Nuclear Information System (INIS)

    Georgieva, Dessislava; Kardas, Anna; Buck, Friedrich; Perbandt, Markus; Betzel, Christian

    2008-01-01

    A novel l-amino-acid oxidase was isolated from V. ammodytes ammodytes venom and crystallized. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. Preliminary diffraction data were collected to 2.6 Å resolution. l-Amino-acid oxidase from the venom of Vipera ammodytes ammodytes, the most venomous snake in Europe, was isolated and crystallized using the sitting-drop vapour-diffusion method. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. The crystals belonged to space group C2, with unit-cell parameters a = 198.37, b = 96.38, c = 109.11 Å, β = 92.56°. Initial diffraction data were collected to 2.6 Å resolution. The calculated Matthews coefficient is approximately 2.6 Å 3 Da −1 assuming the presence of four molecules in the asymmetric unit

  12. Expression, purification, crystallization and preliminary X-ray analysis of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from Helicobacter pylori

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, Paul R.; Mohammad, Shabaz; Melrose, Helen J.; Moody, Peter C. E., E-mail: pcem1@leicester.ac.uk [Henry Wellcome Laboratories for Structural Biology, University of Leicester, Leicester LE1 9HN (United Kingdom)

    2008-08-01

    Glyceraldehyde-3-phosphate dehydrogenase B from H. pylori has been cloned, expressed, purified and crystallized in the presence of NAD. Crystals of GAPDHB diffracted to 2.8 Å resolution and belonged to space group P6{sub 5}22, with unit-cell parameters a = b = 166.1, c = 253.1 Å. Helicobacter pylori is a dangerous human pathogen that resides in the upper gastrointestinal tract. Little is known about its metabolism and with the onset of antibiotic resistance new treatments are required. In this study, the expression, purification, crystallization and preliminary X-ray diffraction of an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from H. pylori are reported.

  13. Crystallization and preliminary X-ray analysis of the flagellar motor `brake' molecule YcgR with c-di-GMP from Escherichia coli.

    Science.gov (United States)

    Hou, Yanjie; Li, De Feng; Wang, Da Cheng

    2013-06-01

    In Escherichia coli and Salmonella enterica, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous bacterial second-messenger molecule that participates in many cellular processes, can regulate flagellar motor speed and reduce cell swimming velocity by binding to the PilZ-containing protein YcgR. Here, the crystallization and preliminary X-ray crystallographic analysis of YcgR with c-di-GMP are reported. The crystals diffracted to 2.3 Å resolution and belonged to space group R3:H, with unit-cell parameters a = b = 93.96, c = 109.61 Å. The asymmetric unit appeared to contain one subunit with a Matthews coefficient of 3.21 Å(3) Da(-1). The results reported here provide a sound basis for solving the crystal structure of YcgR with c-di-GMP and revealing its structure-function relationship based on the three-dimensional structure.

  14. Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

    International Nuclear Information System (INIS)

    Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W.

    2007-01-01

    P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 123.27, c = 134.43 Å

  15. Crystallization and preliminary X-ray diffraction analysis of P30, the transmembrane domain of pertactin, an autotransporter from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yanshi; Black, Isobel; Roszak, Aleksander W.; Isaacs, Neil W., E-mail: n.isaacs@chem.gla.ac.uk [Department of Chemistry and WestChem, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2007-07-01

    P30, the transmembrane C-terminal domain of pertactin from B. pertussis has been crystallized after refolding in vitro. Preliminary X-ray crystallographic data are reported. P30, the 32 kDa transmembrane C-terminal domain of pertactin from Bordetella pertussis, is supposed to form a β-barrel inserted into the outer membrane for the translocation of the passenger domain. P30 was cloned and expressed in inclusion bodies in Escherichia coli. After refolding and purification, the protein was crystallized using the sitting-drop vapour-diffusion method at 292 K. The crystals diffract to a resolution limit of 3.5 Å using synchrotron radiation and belong to the hexagonal space group P6{sub 1}22, with unit-cell parameters a = b = 123.27, c = 134.43 Å.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of the RNA-dependent RNA polymerase from Thosea asigna virus

    International Nuclear Information System (INIS)

    Ferrero, Diego; Buxaderas, Mònica; Rodriguez, José F.; Verdaguer, Núria

    2012-01-01

    The RNA-dependent RNA polymerase of Thosea asigna virus has been purified and crystallized in two different crystal forms. Preliminary characterization of P2 1 2 1 2 and C222 1 crystals is reported. Co-crystallization experiments in the presence of lutetium produced a heavy-atom derivative suitable for structure determination. Thosea asigna virus (TaV) is a positive-sense, single-stranded RNA (ssRNA) virus that belongs to the Permutotetravirus genera within the recently created Permutotetraviridae family. The genome of TaV consists of an RNA segment of about 5.700 nucleotides with two open reading frames, encoding for the replicase and capsid protein. The particular TaV replicase does not contain N7-methyl transferase and helicase domains but includes a structurally unique RNA-dependent RNA polymerase (RdRp) with a sequence permutation in the domain where the active site is anchored. This architecture is also found in double-stranded RNA viruses of the Birnaviridae family. Here we report the purification and preliminary crystallographic studies TaV RdRp. The enzyme was crystallized by the sitting-drop vapour diffusion method using PEG 8K and lithium sulfate as precipitants. Two different crystal forms were obtained: native RdRp crystallized in space group P2 1 2 1 2 and diffracts up to 2.1 Å and the RdRp-Lu 3+ derivative co-crystals belong to the C222 1 space group, diffracting to 3.0 Å resolution. The structure of TaV RdRp represents the first structure of a non-canonical RdRp from ssRNA viruses

  17. Expression, purification, crystallization and preliminary X-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin

    International Nuclear Information System (INIS)

    Hillier, Brian J.; Sundaresan, Vidyasankar; Stout, C. David; Vacquier, Victor D.

    2005-01-01

    The olfactomedin (OLF) domain from the sea urchin cell-adhesion protein amassin has been crystallized. A native data set extending to 2.7 Å has been collected using an in-house X-ray source. A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein–protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 Å under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain

  18. Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

    International Nuclear Information System (INIS)

    Abskharon, Romany N. N.; Soror, Sameh H.; Pardon, Els; El Hassan, Hassan; Legname, Giuseppe; Steyaert, Jan; Wohlkonig, Alexandre

    2010-01-01

    The crystallization of a specific nanobody against mouse PrP C and preliminary diffraction analysis of a crystal that diffracted to 1.23 Å resolution are presented. Prion disorders are infectious diseases that are characterized by the conversion of the cellular prion protein PrP C into the pathogenic isoform PrP Sc . Specific antibodies that interact with the cellular prion protein have been shown to inhibit this transition. Recombinant VHHs (variable domain of dromedary heavy-chain antibodies) or nanobodies are single-domain antibodies, making them the smallest antigen-binding fragments. A specific nanobody (Nb-PrP-01) was raised against mouse PrP C . A crystallization condition for this recombinant nanobody was identified using high-throughput screening. The crystals were optimized using streak-seeding and the hanging-drop method. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 30.04, b = 37.15, c = 83.00 Å, and diffracted to 1.23 Å resolution using synchrotron radiation. The crystal structure of this specific nanobody against PrP C together with the known PrP C structure may help in understanding the PrP C /PrP Sc transition mechanism

  19. Crystallization and preliminary X-ray diffraction analysis of the peptidylprolyl isomerase Par27 of Bordetella pertussis

    International Nuclear Information System (INIS)

    Wohlkönig, Alexandre; Hodak, Hélène; Clantin, Bernard; Sénéchal, Magalie; Bompard, Coralie; Jacob-Dubuisson, Françoise; Villeret, Vincent

    2008-01-01

    Par27 from B. pertussis, the prototype of a new group of parvulins has been crystallized in two different crystal forms. Proteins with both peptidylprolyl isomerase (PPIase) and chaperone activities play a crucial role in protein folding in the periplasm of Gram-negative bacteria. Few such proteins have been structurally characterized and to date only the crystal structure of SurA from Escherichia coli has been reported. Par27, the prototype of a new group of parvulins, has recently been identified. Par27 exhibits both chaperone and PPIase activities in vitro and is the first identified parvulin protein that forms dimers in solution. Par27 has been expressed in E. coli. The protein was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Form A, which belongs to space group P2 (unit-cell parameters a = 42.2, b = 142.8, c = 56.0 Å, β = 95.1°), diffracts to 2.8 Å resolution, while form B, which belongs to space group C222 (unit-cell parameters a = 54.6, b = 214.1, c = 57.8 Å), diffracts to 2.2 Å resolution. Preliminary diffraction data analysis agreed with the presence of one monomer in the asymmetric unit of the orthorhombic crystal form and two in the monoclinic form

  20. Crystallization and preliminary X-ray characterization of a ferritin from the hyperthermophilic archaeon and anaerobe Pyrococcus furiosus

    International Nuclear Information System (INIS)

    Matias, Pedro M.; Tatur, Jana; Carrondo, Maria Arménia; Hagen, Wilfred R.

    2005-01-01

    Ferritin from P. furiosus crystallizes in space group C222 1 , with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å and 36 monomers in the asymmetric unit, corresponding to one and a half 24-mers. Crystals of the title protein have been produced and preliminary structural analysis has been carried out. The crystals belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å. The protein forms a 24-mer of 20 kDa subunits, which assemble with 432 non-crystallographic symmetry. A total of 36 monomers are found in the asymmetric unit, corresponding to one and a half 24-mers

  1. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    International Nuclear Information System (INIS)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-01-01

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K 2 ) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222 1 , with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

  2. Purification, crystallization and preliminary X-ray diffraction studies to near-atomic resolution of dihydrodipicolinate synthase from methicillin-resistant Staphylococcus aureus

    International Nuclear Information System (INIS)

    Burgess, Benjamin R.; Dobson, Renwick C. J.; Dogovski, Con; Jameson, Geoffrey B.; Parker, Michael W.; Perugini, Matthew A.

    2008-01-01

    Dihydrodipicolinate synthase (DHDPS), an enzyme of the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the expression, purification, crystallization and preliminary diffraction analysis to 1.45 Å resolution of DHDPS from methicillin-resistant S. aureus is reported. In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. DHDPS is part of the diaminopimelate pathway leading to lysine, coupling (S)-aspartate-β-semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from methicillin-resistant Staphylococcus aureus, an important bacterial pathogen, are reported. The enzyme was crystallized in a number of forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.45 Å resolution. The space group was P1 and the unit-cell parameters were a = 65.4, b = 67.6, c = 78.0 Å, α = 90.1, β = 68.9, γ = 72.3°. The crystal volume per protein weight (V M ) was 2.34 Å 3 Da −1 , with an estimated solvent content of 47% for four monomers per asymmetric unit. The structure of the enzyme will help to guide the design of novel therapeutics against the methicillin-resistant S. aureus pathogen

  3. Purification, crystallization and preliminary X-ray diffraction experiments on the breakage-reunion domain of the DNA gyrase from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Piton, Jérémie; Matrat, Stéphanie; Petrella, Stéphanie; Jarlier, Vincent; Aubry, Alexandra; Mayer, Claudine

    2009-01-01

    The breakage-reunion domain of M. tuberculosis DNA gyrase was crystallized using the hanging-drop vapour-diffusion method. One of the four crystal forms obtained belonged to space group C2 and diffraction data were collected to a resolution of 2.7 Å. Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57 kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7 Å depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase

  4. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of intracellular growth locus E (IglE) protein from Francisella tularensis subsp. novicida

    International Nuclear Information System (INIS)

    Robb, Craig S.; Nano, Francis E.; Boraston, Alisdair B.

    2010-01-01

    The F. tularensis protein IglE from the FPI, which is a component of the type VI-like secretion system, has been crystallized and preliminary X-ray data have been collected. Tularaemia is an uncommon but potentially dangerous zoonotic disease caused by the bacterium Francisella tularensis. As few as ten bacterial cells are sufficient to cause disease in a healthy human, making this one of the most infectious disease agents known. The virulence of this organism is dependent upon a genetic locus known as the Francisella pathogenicity island (FPI), which encodes components of a secretion system that is related to the type VI secretion system. Here, the cloning, expression, purification and preliminary X-ray diffraction statistics of the FPI-encoded protein IglE are presented. This putative lipoprotein is required for intra-macrophage growth and is thought to be a constituent of the periplasmic portion of the type VI-like protein complex that is responsible for the secretion of critical virulence factors in Francisella

  5. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the variable lymphocyte receptor 2913 ectodomain fused with internalin B

    International Nuclear Information System (INIS)

    Lee, Ji Yeon; Kim, Hyoun Sook; Baek, In Wha; Back, Jang Mi; Han, Mi Ra; Kong, Sun-Young; Kim, Ji Hyeon; Kirchdoerfer, Robert N.; Kim, Jae-Ouk; Cooper, Max D.; Wilson, Ian A.; Kim, Hyun-Jung; Han, Byung Woo

    2012-01-01

    The VLR2913 ectodomain fused with internalin B was crystallized and diffraction data were collected to a maximum resolution of 2.04 Å. In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in the recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, the antigen of which is not known, contains the same HVI amino-acid sequence as that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, identification of the antigen for VLR2913 and the main contributing factors for antigen recognition based on a comparison of VLR2913 and VLR RBC36 has been attempted. To initiate and facilitate this structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of internalin B (InlB-VLR2913-ECD). Three amino-acid residues on the concave surface of the LRR modules of InlB-VLR2913-ECD were mutated, considering important residues for hydrogen bonds in the recognition of H-trisaccharide by VLR RBC36. InlB-VLR2913-ECD was overexpressed in Escherichia coli and was crystallized at 295 K using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.04 Å resolution and could be indexed in the tetragonal space group P4 1 2 1 2 (or P4 3 2 1 2), with unit-cell parameters a = 91.12, b = 91.12, c = 62.87 Å. Assuming that one monomer molecule was present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V M ) was 2.75 Å 3 Da −1 and the solvent content was 55.2%. Structural determination of InlB-VLR2913-ECD by molecular replacement is in progress

  6. Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4.

    Science.gov (United States)

    Xu, Xiang; Zhang, Xiaoai; Lu, Guangwen; Cai, Yongping

    2012-08-01

    Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, β = 94.7°. Preliminary analysis of the diffraction data was also performed.

  7. Overproduction, crystallization and preliminary X-ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli

    International Nuclear Information System (INIS)

    Garces, Fernando; Fernández, Francisco J.; Pérez-Luque, Rosa; Aguilar, Juan; Baldomà, Laura; Coll, Miquel; Badía, Josefa; Vega, M. Cristina

    2007-01-01

    UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K. UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å 3 Da −1 and a solvent content of 43%

  8. Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

    International Nuclear Information System (INIS)

    Voss, Jarrod E.; Scally, Stephen W.; Taylor, Nicole L.; Dogovski, Con; Alderton, Malcolm R.; Hutton, Craig A.; Gerrard, Juliet A.; Parker, Michael W.; Dobson, Renwick C. J.; Perugini, Matthew A.

    2009-01-01

    Dihydrodipicolinate synthase (DHDPS) catalyses an important step in lysine biosynthesis. Here, the expression, purification, crystallization and preliminary diffraction analysis to 2.15 Å resolution of DHDPS from B. anthracis soaked with the substrate pyruvate are reported. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the lysine-biosynthesis pathway in bacteria, plants and some fungi. In this study, the expression of DHDPS from Bacillus anthracis (Ba-DHDPS) and the purification of the recombinant enzyme in the absence and presence of the substrate pyruvate are described. It is shown that DHDPS from B. anthracis purified in the presence of pyruvate yields greater amounts of recombinant enzyme with more than 20-fold greater specific activity compared with the enzyme purified in the absence of substrate. It was therefore sought to crystallize Ba-DHDPS in the presence of the substrate. Pyruvate was soaked into crystals of Ba-DHDPS prepared in 0.2 M sodium fluoride, 20%(w/v) PEG 3350 and 0.1 M bis-tris propane pH 8.0. Preliminary X-ray diffraction data of the recombinant enzyme soaked with pyruvate at a resolution of 2.15 Å are presented. The pending crystal structure of the pyruvate-bound form of Ba-DHDPS will provide insight into the function and stability of this essential bacterial enzyme

  9. Crystallization and preliminary X-ray characterization of the glpX-encoded class II fructose-1,6-bisphosphatase from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Gutka, Hiten J.; Franzblau, Scott G.; Movahedzadeh, Farahnaz; Abad-Zapatero, Cele

    2011-01-01

    The crystallization and preliminary X-ray crystallographic analysis of the glpX-encoded class II fructose-1,6-bisphosphatase from M. tuberculosis in the apo form is reported. Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Å and belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 131.3, c = 143.2 Å. The structure has been solved by molecular replacement and is currently undergoing refinement

  10. Crystallization, characterization and preliminary X-ray crystallographic analysis of GK2848, a putative carbonic anhydrase of Geobacillus kaustophilus

    International Nuclear Information System (INIS)

    Ragunathan, Preethi; Raghunath, Gokul; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2013-01-01

    The expression, purification, characterization and crystallization of GK2848, a carbonic anhydrase from G. kaustophilus, are described. The crystals diffracted to a resolution of 2.70 Å. GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni–NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2 1 2 1 2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur–Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized

  11. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of molybdopterin synthase from Thermus thermophilus HB8

    International Nuclear Information System (INIS)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Ohmori, Miwa; Agari, Kazuko; Kitamura, Yoshiaki; Baba, Seiki; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2007-01-01

    The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P2 1 and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation. The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

  12. Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment

    International Nuclear Information System (INIS)

    Sugiyama, Shigeru; Nomura, Yusuke; Sakamoto, Taiichi; Kitatani, Tomoya; Kobayashi, Asako; Miyakawa, Shin; Takahashi, Yoshinori; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Nakamura, Yoshikazu; Matsumura, Hiroyoshi

    2008-01-01

    An RNA aptamer in complex with the human IgG Fc fragment have been crystallized. The stirring technique with a rotary shaker was used to improve the crystals and to ensure that they were of high quality and single, resulting in crystals that diffracted to 2.2 Å resolution. Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution

  13. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of succinyl-diaminopimelate desuccinylase (Rv1202, DapE) from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Reinhard, Linda; Mueller-Dieckmann, Jochen; Weiss, Manfred S.

    2012-01-01

    M. tuberculosis succinyl-diaminopimelate desuccinylase, the enzyme which catalyzes the seventh step of the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized. Preliminary X-ray diffraction analysis indicated the presence of pseudo-merohedral twinning in space group P2 1 , resulting in possible emulation of space group C222 1 . Succinyl-diaminopimelate desuccinylase from Mycobacterium tuberculosis (DapE, Rv1202) has been cloned, heterologously expressed in Escherichia coli and purified using standard chromatographic techniques. Diffraction-quality crystals were obtained at acidic pH from ammonium sulfate and PEG and diffraction data were collected from two crystals to resolutions of 2.40 and 2.58 Å, respectively. The crystals belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 79.7, b = 76.0, c = 82.9 Å, β = 119°. The most probable content of the asymmetric unit was two molecules of DapE, which would correspond to a solvent content of 56%. Both examined crystals turned out to be pseudo-merohedrally twinned, with twin operator −h, −k, h + l and twin fractions of approximately 0.46 and 0.16, respectively

  14. Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A2 complexed with caffeic acid, a molecule with inhibitory properties against snake venoms

    International Nuclear Information System (INIS)

    Shimabuku, Patrícia S.; Fernandes, Carlos A. H.; Magro, Angelo J.; Costa, Tássia R.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    Piratoxin I, a noncatalytic and myotoxic Lys49-phospholipase A 2 from B. pirajai venom, was cocrystallized with the inhibitor caffeic acid and a data set was collected to a resolution of 1.65 Å. The electron-density map unambiguously indicated that three inhibitor molecules interact with the C-terminus of the protein. Phospholipases A 2 (PLA 2 s) are one of the main components of bothropic venoms; in addition to their phospholipid hydrolysis action, they are involved in a wide spectrum of pharmacological activities, including neurotoxicity, myotoxicity and cardiotoxicity. Caffeic acid is an inhibitor that is present in several plants and is employed for the treatment of ophidian envenomations in the folk medicine of many developing countries; as bothropic snake bites are not efficiently neutralized by conventional serum therapy, it may be useful as an antivenom. In this work, the cocrystallization and preliminary X-ray diffraction analysis of the Lys49-PLA 2 piratoxin I from Bothrops pirajai venom in the presence of the inhibitor caffeic acid (CA) are reported. The crystals diffracted X-rays to 1.65 Å resolution and the structure was solved by molecular-replacement techniques. The electron-density map unambiguously indicated the presence of three CA molecules that interact with the C-terminus of the protein. This is the first time a ligand has been observed bound to this region and is in agreement with various experiments previously reported in the literature

  15. Purification, crystallization and preliminary X-ray analysis of the IgV domain of human nectin-4

    International Nuclear Information System (INIS)

    Xu, Xiang; Zhang, Xiaoai; Lu, Guangwen; Cai, Yongping

    2012-01-01

    Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported Nectin-4 belongs to a family of immunoglobulin-like cell adhesion molecules and is highly expressed in cancer cells. Recently, nectin-4 was found to be a receptor of measles virus and the IgV domain sustains strong binding to measles virus H protein. In this study, the successful expression and purification of human nectin-4 V domain (nectin-4v) is reported. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.8 Å resolution and belonged to space group P2 1 , with unit-cell parameters a = 33.1, b = 51.7, c = 56.9 Å, β = 94.7°. Preliminary analysis of the diffraction data was also performed

  16. Cloning, expression, crystallization and preliminary X-ray analysis of a putative multiple antibiotic resistance repressor protein (MarR) from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Tu, Zhi-Le; Li, Juo-Ning; Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Gao, Fei Philip; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A putative repressor for the multiple antibiotic resistance operon from a plant pathogen X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. The crystals diffracted to 2.3 Å with good quality. The multiple antibiotic resistance operon (marRAB) is a member of the multidrug-resistance system. When induced, this operon enhances resistance of bacteria to a variety of medically important antibiotics, causing a serious global health problem. MarR is a marR-encoded protein that represses the transcription of the marRAB operon. Through binding with salicylate and certain antibiotics, however, MarR can derepress and activate the marRAB operon. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC1739, a putative MarR repressor protein present in the Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, are described. The XC1739 crystals diffracted to a resolution of at least 1.8 Å. They are orthorhombic and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 39.5, b = 54.2 and c = 139.5 Å, respectively. They contain two molecules in the asymmetric unit from calculation of the self-rotation function

  17. Expression, crystallization and preliminary X-ray data analysis of NT-Als9-2, a fungal adhesin from Candida albicans

    International Nuclear Information System (INIS)

    Salgado, Paula S.; Yan, Robert; Rowan, Fiona; Cota, Ernesto

    2011-01-01

    Details of the expression and crystallization of the N-terminal fragment of Als9-2, an adhesin from the human commensal/pathogenic fungus C. albicans, are reported. Preliminary analysis of the collected X-ray data is also discussed. Candida albicans is a common human fungal commensal that can also cause a range of infections from skin/mucosal ‘thrush’ to severe systemic candidiasis. Adherence to host cells is one of the key determinants of Candida pathogenesis. The Als family of surface proteins has been implicated in adhesion of C. albicans, yet limited information has been published on the structure and mechanism of these fungal adhesins. The N-terminal region of these proteins has been shown to possess adhesive properties, making it a possible target for new therapeutic strategies. Recombinant NT-Als9-2 from C. albicans (residues 18–329) was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.0 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 34.73, b = 68.71, c = 120.03 Å, α = β = γ = 90° and one molecule in the asymmetric unit. Platinum-derivatized crystals belonged to the same space group, with similar unit-cell parameters, although they were not completely isomorphous

  18. Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis

    International Nuclear Information System (INIS)

    Lin, Chien-Hung; Chin, Ko-Hsin; Gao, Fei Philip; Lyu, Ping-Chiang; Shr, Hui-Lin; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-01-01

    A probable copper-ion tolerance protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 Å. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 Å

  19. Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, d-alanine-d-alanine ligase A, from Xanthomonas oryzae pv. oryzae

    International Nuclear Information System (INIS)

    Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Kim, Hyesoon; Ahn, Yeh-Jin; Kim, Jeong-Gu; Lee, Byoung-Moo; Kang, Lin-Woo

    2008-01-01

    Xoo0352, which encodes d-alanine-d-alanine ligase A (DdlA), from X. oryzae pv. oryzae was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of DdlA crystals was performed. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. d-Alanine-d-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in d-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of d-alanyl-d-alanine from two d-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 Å resolution and belonged to the primitive tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 83.0, c = 97.6 Å. There is one molecule in the asymmetric unit, with a corresponding V M of 1.88 Å 3 Da −1 and a solvent content of 34.6%. The initial structure was determined by molecular replacement using d-alanine-d-alanine ligase from Staphylococcus aureus as a template model

  20. Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

    International Nuclear Information System (INIS)

    Cicmil, Nenad

    2008-01-01

    T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N 5 ,N 10 -methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å

  1. Crystallization and preliminary X-ray crystallographic characterization of TrmFO, a folate-dependent tRNA methyltransferase from Thermotoga maritima

    Energy Technology Data Exchange (ETDEWEB)

    Cicmil, Nenad, E-mail: cicmil@uiuc.edu [Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

    2008-03-01

    T. maritima TrmFO was overexpressed, purified and crystallized. A diffraction data set was collected to a resolution of 2.6 Å. TrmFO, previously classified as GID, is a methyltransferase that catalyzes the formation of 5-methyluridine or ribothymidine (T) at position 54 in tRNA in some Gram-positive bacteria. To date, TrmFO is the only characterized tRNA methyltransferase that does not use S-adenosylmethionine as the methyl-group donor. Instead, the donor of the methyl group is N{sup 5},N{sup 10}-methylenetetrahydrofolate. The crystallization and preliminary X-ray crystallographic studies of TrmFO are reported here. The recombinant protein, cloned from Thermotoga maritima genomic DNA, was overproduced in Esherichia coli and crystallized in 25%(v/v) PEG 4000, 100 mM NaCl and sodium citrate buffer pH 5.0 at 291 K using the hanging-drop vapor-diffusion method. The plate-shaped crystals diffracted to 2.6 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 79.94, b = 92.46, c = 127.20 Å.

  2. Crystallization and preliminary X-ray diffraction analysis of two extracytoplasmic solute receptors of the DctP family from Bordetella pertussis

    International Nuclear Information System (INIS)

    Rucktooa, Prakash; Huvent, Isabelle; Antoine, Rudy; Lecher, Sophie; Jacob-Dubuisson, Françoise; Villeret, Vincent; Bompard, Coralie

    2006-01-01

    Sample preparation, crystallization and preliminary X-ray analysis are reported for two B. pertussis extracytoplasmic solute receptors. DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P4 1 2 1 2, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 Å, while selenomethionyl-derivatized DctP7 crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 Å. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods

  3. Crystallization and preliminary X-ray diffraction analysis of two extracytoplasmic solute receptors of the DctP family from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Rucktooa, Prakash; Huvent, Isabelle [UMR8161 CNRS Institut de Biologie de Lille, Laboratoire de Cristallographie Macromoléculaire, 1 Rue du Professeur Calmette, BP 447, 59021 Lille CEDEX (France); IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); Antoine, Rudy; Lecher, Sophie; Jacob-Dubuisson, Françoise, E-mail: francoise.jacob@ibl.fr [IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); INSERM-U629, Lille (France); Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France); Villeret, Vincent, E-mail: francoise.jacob@ibl.fr; Bompard, Coralie [UMR8161 CNRS Institut de Biologie de Lille, Laboratoire de Cristallographie Macromoléculaire, 1 Rue du Professeur Calmette, BP 447, 59021 Lille CEDEX (France); IFR 142, Institut Pasteur de Lille, 1 Rue du Professeur Calmette, BP 245, 59021 Lille CEDEX (France)

    2006-10-01

    Sample preparation, crystallization and preliminary X-ray analysis are reported for two B. pertussis extracytoplasmic solute receptors. DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 Å, while selenomethionyl-derivatized DctP7 crystallized in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 Å. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods.

  4. Crystallization and preliminary X-ray diffraction study of phosphopantetheine adenylyltransferase from M. tuberculosis crystallizing in space group P3{sub 2}

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Chupova, L. A.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-09-15

    Crystals of M. tuberculosis phosphopantetheine adenylyltransferase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 2.00-Å resolution. The crystals belong to sp. gr. P3{sub 2} and have the following unit-cell parameters: a = b = 106.47 Å, c = 71.32 Å, α = γ = 90°, β = 120°. The structure was solved by the molecular-replacement method. There are six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is a biologically active form of phosphopantetheine adenylyltransferase from M. tuberculosis.

  5. Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Prem [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India); Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh [Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012 (India); Vijayan, M., E-mail: mv@mbu.iisc.ernet.in [Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 (India)

    2006-12-01

    Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

  6. Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

    International Nuclear Information System (INIS)

    Singh, Prem; Talawar, Ramappa K.; Krishna, P. D. V.; Varshney, Umesh; Vijayan, M.

    2006-01-01

    Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected. Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis

  7. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708

    OpenAIRE

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    Conjugated polyketone reductase C2 from C. parapsilosis IFO 0708 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group P212121 and diffracted X-rays to 1.7 Å resolution.

  8. Human tRNAGly acceptor-stem microhelix: crystallization and preliminary X-ray diffraction analysis at 1.2 Å resolution

    International Nuclear Information System (INIS)

    Förster, Charlotte; Szkaradkiewicz, Karol; Perbandt, Markus; Brauer, Arnd B. E.; Borowski, Tordis; Fürste, Jens P.; Betzel, Christian; Erdmann, Volker A.

    2007-01-01

    The human tRNA Gly acceptor-stem microhelix was crystallized and preliminary X-ray diffraction analysis revealed diffraction to a resolution of up to 1.2 Å. The major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-tRNA synthetase systems (GlyRS; class II aminoacyl-tRNA synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. The differences in the identity elements of the respective tRNA Gly systems are located within the acceptor stem and include the discriminator base U73. In the present work, the human tRNA Gly acceptor-stem microhelix was crystallized in an attempt to analyze the structural features that govern the correct recognition of tRNA Gly by the eukaryotic/archaebacterial-type glycyl-tRNA synthetase. The crystals of the human tRNA Gly acceptor-stem helix belong to the monoclinic space group C2, with unit-cell parameters a = 37.12, b = 37.49, c = 30.38 Å, α = γ = 90, β = 113.02°, and contain one molecule per asymmetric unit. A high-resolution data set was acquired using synchrotron radiation and the data were processed to 1.2 Å resolution

  9. Crystallization and preliminary X-ray crystallographic investigations on a βγ-crystallin domain of absent in melanoma 1 (AIM1), a protein from Homo sapiens

    International Nuclear Information System (INIS)

    Aravind, Penmatsa; Rajini, Bheemreddy; Sharma, Yogendra; Sankaranarayanan, Rajan

    2006-01-01

    The crystallization and preliminary X-ray diffraction analysis of AIM1g1, a βγ-crystallin domain of absent in melanoma (AIM1) protein from H. sapiens, is reported. AIM1g1 is a single βγ-crystallin domain from the protein absent in melanoma 1 (AIM1), which appears to play a role in the suppression of melanomas. This domain is known to bind calcium and its structure would help in identifying calcium-coordinating sites in vertebrate crystallins, which have hitherto been believed to have lost this ability during evolution. Crystallization of this domain was performed by the hanging-drop vapour-diffusion method. Crystals diffracted to a maximum resolution of 1.86 Å and were found to belong to space group P6 1 or P6 5 , with unit-cell parameters a = b = 54.98, c = 59.73 Å. Solvent-content analysis indicated the presence of one monomer per asymmetric unit

  10. Crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of the human spliceosomal DExD/H-box protein hPrp22

    International Nuclear Information System (INIS)

    Kudlinzki, Denis; Nagel, Christian; Ficner, Ralf

    2009-01-01

    The cloning, purification and crystallization of the C-terminal domain of human hPrp22 are reported. This communication also contains data for the preliminary X-ray diffraction analysis. The Homo sapiens DExD/H-box protein hPrp22 is a crucial component of the eukaryotic pre-mRNA splicing machinery. Within the splicing cycle, it is involved in the ligation of exons and generation of the lariat and it additionally catalyzes the release of mature mRNA from the spliceosomal U5 snRNP. The yeast homologue of this protein, yPrp22, shows ATP-dependent RNA-helicase activity and is capable of unwinding RNA/RNA duplex molecules. A truncated construct coding for residues 950–1183 of human Prp22, comprising the structurally and functionally uncharacterized C-terminal domain, was cloned into an Escherichia coli expression vector. The protein was subsequently overproduced, purified and crystallized. The crystals obtained diffracted to 2.1 Å resolution, belonged to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = b = 78.2, c = 88.4 Å, and contained one molecule in the asymmetric unit

  11. Crystallization and preliminary X-ray crystallographic investigations on a βγ-crystallin domain of absent in melanoma 1 (AIM1), a protein from Homo sapiens

    Energy Technology Data Exchange (ETDEWEB)

    Aravind, Penmatsa; Rajini, Bheemreddy; Sharma, Yogendra; Sankaranarayanan, Rajan, E-mail: sankar@ccmb.res.in [Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007 (India)

    2006-03-01

    The crystallization and preliminary X-ray diffraction analysis of AIM1g1, a βγ-crystallin domain of absent in melanoma (AIM1) protein from H. sapiens, is reported. AIM1g1 is a single βγ-crystallin domain from the protein absent in melanoma 1 (AIM1), which appears to play a role in the suppression of melanomas. This domain is known to bind calcium and its structure would help in identifying calcium-coordinating sites in vertebrate crystallins, which have hitherto been believed to have lost this ability during evolution. Crystallization of this domain was performed by the hanging-drop vapour-diffusion method. Crystals diffracted to a maximum resolution of 1.86 Å and were found to belong to space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 54.98, c = 59.73 Å. Solvent-content analysis indicated the presence of one monomer per asymmetric unit.

  12. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    International Nuclear Information System (INIS)

    Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

    2010-01-01

    Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

  13. Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila

    International Nuclear Information System (INIS)

    Varshney, Nishant Kumar; Ramasamy, Sureshkumar; Brannigan, James A.; Wilkinson, Anthony J.; Suresh, C. G.

    2013-01-01

    The pac gene encoding penicillin G acylase from K. citrophila was cloned and a slow-processing site-directed mutant was prepared, expressed, purified and crystallized. Triclinic and monoclinic crystal forms were obtained which diffracted to 2.5 and 3.5 Å resolution, respectively. Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide

  14. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    International Nuclear Information System (INIS)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru

    2007-01-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2 1 2 1 2 1 , with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V M = 2.4 Å 3 Da −1 ) and had a solvent content of 48%

  15. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2007-06-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V{sub M} = 2.4 Å{sup 3} Da{sup −1}) and had a solvent content of 48%.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A2 with vasoconstrictor activity from Agkistrodon halys pallas venom

    International Nuclear Information System (INIS)

    Zou, Zhisong; Zeng, Fuxing; Zhang, Lu; Niu, Liwen; Teng, Maikun; Li, Xu

    2012-01-01

    A vasoconstrictor PLA 2 was purified from Agkistrodon halys pallas venom and the preliminary X-ray diffraction analysis had been described. Phospholipases A 2 (PLA 2 s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA 2 , here named AhV-aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV-aPA migrated as a single band on SDS–PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA 2 s, AhV-aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV-aPA could induce a further contractile response on the 60 mM K + -induced contraction, with an EC 50 of 369 nmol l −1 . Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a = 44.27, b = 68.39, c = 81.54 Å

  17. Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex

    Energy Technology Data Exchange (ETDEWEB)

    Huet, Joëlle, E-mail: jhuet@ulb.ac.be [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium); Azarkan, Mohamed [Laboratoire de Chimie Générale (CP 609), Faculté de Médecine, Université Libre de Bruxelles (ULB), Campus Erasme, 808 Route de Lennik, B-1070 Bruxelles (Belgium); Looze, Yvan [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium); Villeret, Vincent [CNRS-UMR 8161, Institut de Biologie de Lille, Université de Lille 1-Université de Lille 2-Institut Pasteur de Lille, IFR142, 1 Rue du Professeur Calmette, F-59021 Lille (France); Wintjens, René, E-mail: jhuet@ulb.ac.be [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium)

    2008-05-01

    A chitinase isolated from the latex of the tropical species Carica papaya has been crystallized. The addition of N-acetyl-d-glucosamine to the crystallization solution has improved the diffraction quality resolution of the crystal to 1.8 Å resolution. A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 Å, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-d-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 Å, β = 95.33° and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 Å. Structure refinement is currently in progress.

  18. Crystallization and preliminary X-ray analysis of formate oxidase, an enzyme of the glucose–methanol–choline oxidoreductase family

    International Nuclear Information System (INIS)

    Maeda, Yoshifumi; Doubayashi, Daiju; Ootake, Takumi; Oki, Masaya; Mikami, Bunzo; Uchida, Hiroyuki

    2010-01-01

    Formate oxidase from A. oryzae RIB40 was crystallized and diffraction data were collected to a resolution of 2.4 Å. Formate oxidase (FOD), which catalyzes the oxidation of formate to yield carbon dioxide and hydrogen peroxide, belongs to the glucose–methanol–choline oxidoreductase (GMCO) family. FOD from Aspergillus oryzae RIB40, which has a modified FAD as a cofactor, was crystallized at 293 K by the hanging-drop vapour-diffusion method. The crystal was orthorhombic and belonged to space group C222 1 . Diffraction data were collected from a single crystal to 2.4 Å resolution

  19. Perfect-crystal x-ray optics to treat x-ray coherence

    International Nuclear Information System (INIS)

    Yamazaki, Hiroshi; Ishikawa, Tetsuya

    2007-01-01

    X-ray diffraction of perfect crystals, which serve as x-ray monochromator and collimator, modifies coherence properties of x-ray beams. From the time-dependent Takagi-Taupin equations that x-ray wavefields obey in crystals, the reflected wavefield is formulated as an integral transform of a general incident wavefield with temporal and spatial inhomogeneity. A reformulation of rocking-curve profiles from the field solution of the Takagi-Taupin equations allows experimental evaluation of the mutual coherence function of x-ray beam. The rigorous relationship of the coherence functions between before and after reflection clarifies how the coherence is transferred by a crystal. These results will be beneficial to developers of beamline optics for the next generation synchrotron sources. (author)

  20. Purification, crystallization and preliminary X-ray diffraction of the G3BP1 NTF2-like domain

    DEFF Research Database (Denmark)

    Vognsen, Tina; Möller, Ingvar Rúnar; Kristensen, Ole

    2011-01-01

    The nuclear transport factor 2-like (NTF2-like) domain of human G3BP1 was subcloned, overexpressed in Escherichia coli and purified. Crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected to 3.6 Å resolution using synchrotron radiation. The crystals...

  1. Cloning, expression, purification, crystallization and preliminary X-ray analysis of Thermus aquaticus succinyl-CoA synthetase

    International Nuclear Information System (INIS)

    Joyce, Michael A.; Brownie, Edward R.; Hayakawa, Koto; Fraser, Marie E.

    2007-01-01

    Attempts to crystallize succinyl-CoA synthetase from the thermophile T. aquaticus were thwarted by proteolysis of the β-subunit and preferential crystallization of a truncated form. Crystals of the full-length enzyme were grown after the purification protocol was modified to include frequent additions of protease inhibitors. Succinyl-CoA synthetase (SCS) is an enzyme of the citric acid cycle and is thus found in most species. To date, there are no structures available of SCS from a thermophilic organism. To investigate how the enzyme adapts to higher temperatures, SCS from Thermus aquaticus was cloned, overexpressed, purified and crystallized. Attempts to crystallize the enzyme were thwarted by proteolysis of the β-subunit and preferential crystallization of the truncated form. Crystals of full-length SCS were grown after the purification protocol was modified to include frequent additions of protease inhibitors. The resulting crystals, which diffract to 2.35 Å resolution, are of the protein in complex with Mn 2+ -GDP

  2. Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Dobritzsch, D.; Gojkovic, Zoran; Andersen, Birgit

    2003-01-01

    In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2...

  3. Crystallization and preliminary X-ray analysis of the bacillaene synthase trans-acting acyltransferase PksC

    International Nuclear Information System (INIS)

    Cuskin, Fiona; Solovyova, Alexandra S.; Lewis, Richard J.; Race, Paul R.

    2011-01-01

    The expression, purification and crystallization of the trans-acting acyltransferase PksC from the bacillaene hybrid polyketide synthase/nonribosomal peptide synthetase is described. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 and diffracted to 1.44 Å resolution. The antibiotic bacillaene is biosynthesized in Bacillus subtilis by a hybrid type 1 modular polyketide synthase/nonribosomal peptide synthetase of the trans-acyltransferase (trans-AT) class. Within this system, the essential acyl-group loading activity is provided by the action of three free-standing trans-acting acyltransferases. Here, the recombinant expression, purification and crystallization of the bacillaene synthase trans-acting acyltransferase PksC are reported. A diffraction data set has been collected from a single PksC crystal to 1.44 Å resolution and the crystal was found to belong to the orthorhombic space group P2 1 2 1 2 1

  4. Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

    International Nuclear Information System (INIS)

    Suzuki, Ryuichiro; Kim, Byung-Jun; Shibata, Tsuyoshi; Iwamoto, Yuki; Katayama, Takane; Ashida, Hisashi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya; Yamamoto, Kenji

    2010-01-01

    Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl 2 , 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

  5. Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase ATPase domain

    International Nuclear Information System (INIS)

    Roué, Mélanie; Agrawal, Alka; Volker, Craig; Mossakowska, Danuta; Mayer, Claudine; Bax, Benjamin D.

    2013-01-01

    The ATPase domain of M. tuberculosis DNA gyrase was crystallized using hanging-drop vapour diffusion. The crystals belonged to space groups P1 and P2 1 . Diffraction data were collected to resolutions of 2.9 and 3.3 Å, respectively. Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47 C1 and Mtb-GyrB47 C2 , have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P2 1 and diffracted to resolutions of 2.9 and 3.3 Å, respectively

  6. Preparation, crystallization, and preliminary X-ray diffraction study of mutant carboxypeptidase T containing the primary specificity pocket of carboxypeptidase B

    International Nuclear Information System (INIS)

    Akparov, V. Kh.; Grishin, A. M.; Timofeev, V. I.; Kuranova, I. P.

    2010-01-01

    Recombinant G215S, A251G, T257A, D260G, T262D mutant carboxypeptidase T from Thermoactinomyces vulgaris containing mutations in the primary specificity pocket was prepared and crystallized. Single crystals with a size of up to 0.3 mm were grown and investigated by X-ray diffraction. Recombinant mutant carboxypeptidase T containing the primary specificity subsite compositionally identical to that of pancreatic carboxypeptidase B crystallizes in the same space group as the natural enzyme. The crystals belong to sp. gr. P6 3 22; the unit-cell parameters are a = b = 157.867 A, c = 104.304 A, α = β = 90 deg., γ = 120 deg. X-ray diffraction data suitable for determining the three-dimensional structure at atomic resolution were collected from one crystal.

  7. Crystallization and preliminary X-ray analysis of Acetivibrio cellulolyticus cellulosomal type II cohesin module: two versions having different linker lengths

    International Nuclear Information System (INIS)

    Noach, Ilit; Alber, Orly; Bayer, Edward A.; Lamed, Raphael; Levy-Assaraf, Maly; Shimon, Linda J. W.; Frolow, Felix

    2007-01-01

    The cloning, expression, purification, crystallization and preliminary X-ray characterization of two protein constructs of the second type II cohesin module from A. cellulolyticus ScaB are described. Both constructs contain the native N-terminal linker, but only one of them contains the full-length 45-residue C-terminal linker; the other contains a five-residue segment of this linker. The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2-S) and the second incorporating the full native 45-residue linker (CohB2-L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2-S and CohB2-L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2 1 2 1 2 1 with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2-S and space group P2 1 2 1 2 with unit-cell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2-L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2-S contains three cohesin molecules, while that of CohB2-L contains two molecules

  8. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum

    International Nuclear Information System (INIS)

    Eckhard, Ulrich; Nüss, Dorota; Ducka, Paulina; Schönauer, Esther; Brandstetter, Hans

    2008-01-01

    The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution. The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%

  9. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum

    Energy Technology Data Exchange (ETDEWEB)

    Eckhard, Ulrich, E-mail: ulrich.eckhard@sbg.ac.at; Nüss, Dorota; Ducka, Paulina; Schönauer, Esther; Brandstetter, Hans [Structural Biology Group, Department of Molecular Biology, University of Salzburg, 5020 Salzburg (Austria)

    2008-05-01

    The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution. The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%.

  10. Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis

    International Nuclear Information System (INIS)

    Tamura, Haruka; Ashida, Hiroki; Koga, Shogo; Saito, Yohtaro; Yadani, Tomonori; Kai, Yasushi; Inoue, Tsuyoshi; Yokota, Akiho; Matsumura, Hiroyoshi

    2009-01-01

    Crystals of the 45.1 kDa functional form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from B. subtilis diffracted to 2.30 Å resolution. 2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P2 1 , with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å 3 Da −1 and a solvent content of 43%

  11. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Hiromi; Yamada, Mitsugu [Molecular Structure Research Group, Information Technology Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793 (Japan); Nishitani, Takeyori; Takada, Goro; Izumori, Ken [Department of Biochemistry and Food Science, Faculty of Agriculture and Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Kamitori, Shigehiro, E-mail: kamitori@med.kagawa-u.ac.jp [Molecular Structure Research Group, Information Technology Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793 (Japan)

    2007-02-01

    Recombinant d-tagatose 3-epimerase from P. cichorii was purified and crystallized. Diffraction data were collected to 2.5 Å resolution. d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-psicose has not been reported with epimerases other than P. cichorii D-TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules.

  12. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    International Nuclear Information System (INIS)

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-01-01

    Recombinant d-tagatose 3-epimerase from P. cichorii was purified and crystallized. Diffraction data were collected to 2.5 Å resolution. d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-psicose has not been reported with epimerases other than P. cichorii D-TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2 1 , with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules

  13. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    Jong, René M. de; Rozeboom, Henriëtte J.; Kalk, Kor H.; Tang, Lixia; Janssen, Dick B.; Dijkstra, Bauke W.

    2002-01-01

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  14. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    de Jong, RM; Rozeboom, HJ; Kalk, KH; Tang, Lixia; Janssen, DB; Dijkstra, BW

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  15. Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

    International Nuclear Information System (INIS)

    Swairjo, Manal A.; Reddy, Robert R.; Lee, Bobby; Van Lanen, Steven G.; Brown, Shannon; Crécy-Lagard, Valérie de; Iwata-Reuyl, Dirk; Schimmel, Paul

    2005-01-01

    Structural informatics and modelling correctly predicted that substrate was required to obtain diffracting crystals of the first characterized nitrile oxidoreductase: the homododecameric QueF. QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 Å. A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ 0 . This crystal form diffracted to 2.25 Å resolution

  16. Crystallization and preliminary X-ray analysis of LipL32 from Leptospira interrogans serovar Copenhageni

    International Nuclear Information System (INIS)

    Hauk, Pricila; Guzzo, Cristiane R.; Ho, Paulo L.; Farah, Chuck S.

    2009-01-01

    Recombinant selenomethionine-labelled LipL32, the major surface protein of pathogenic Leptospira, has been purified and crystallized. Data sets from two crystals were collected, one of which diffracted to 2.25 Å resolution. LipL32 is a major surface protein that is expressed during infection by pathogenic Leptospira. Here, the crystallization of recombinant LipL32 21–272 , which corresponds to the mature LipL32 protein minus its N-terminal lipid-anchored cysteine residue, is described. Selenomethionine-labelled LipL32 21–272 crystals diffracted to 2.25 Å resolution at a synchrotron source. The space group was P3 1 21 or P3 2 21 and the unit-cell parameters were a = b = 126.7, c = 96.0 Å

  17. Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

    International Nuclear Information System (INIS)

    Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

    2007-01-01

    A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3 1 21 or P3 2 21

  18. Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain

    International Nuclear Information System (INIS)

    Roy, Sumana; Choudhury, Debi; Chakrabarti, Chandana; Biswas, Sampa; Dattagupta, J. K.

    2011-01-01

    The crystallization of the precursor of a thermostable variant of papain and the collection of diffraction data to 2.6 Å resolution are reported. The crystallization of a recombinant thermostable variant of pro-papain has been carried out. The mutant pro-enzyme was expressed in Escherichia coli as inclusion bodies, refolded, purified and crystallized. The crystals belonged to space group P2 1 , with unit-cell parameters a = 42.9, b = 74.8, c = 116.5 Å, β = 93.0°, and diffracted to 2.6 Å resolution using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, the calculated Matthews coefficient is 2.28 Å 3 Da −1 , corresponding to a solvent content of 46%. Initial attempts to solve the structure using molecular-replacement techniques were successful

  19. Cloning, expression, purification, crystallization and preliminary X-ray analysis of a fructokinase from Vibrio cholerae O395

    International Nuclear Information System (INIS)

    Paul, Rakhi; Nath, Seema; Sen, Udayaditya

    2012-01-01

    A fructokinase from V. cholerae O395 has been cloned, expressed, purified and crystallized in the apo form; the crystals belonged to the orthorhombic space group P2 1 2 1 2 and diffracted to 2.45 Å resolution. Fructokinase with ADP and fructose bound has also been crystallized and the crystals diffracted to a resolution of 1.75 Å. Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni–NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg 2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P2 1 2 1 2, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4 Å 3 Da −1 , which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1 Å 3 Da −1 and 40%, respectively, assuming the presence of one molecule in the asymmetric unit

  20. Purification, crystallization and preliminary X-ray diffraction analysis of the HMG domain of Sox17 in complex with DNA

    International Nuclear Information System (INIS)

    Ng, Calista Keow Leng; Palasingam, Paaventhan; Venkatachalam, Rajakannan; Baburajendran, Nithya; Cheng, Jason; Jauch, Ralf; Kolatkar, Prasanna R.

    2008-01-01

    Crystals of the Sox17 HMG domain bound to LAMA1 enhancer DNA-element crystals that diffracted to 2.75 Å resolution were obtained. Sox17 is a member of the SRY-related high-mobility group (HMG) of transcription factors that have been shown to direct endodermal differentiation in early mammalian development. The LAMA1 gene encoding the α-chain of laminin-1 has been reported to be directly bound and regulated by Sox17. This paper describes the details of initial crystallization attempts with the HMG domain of mouse Sox17 (mSox17-HMG) with a 16-mer DNA element derived from the LAMA1 enhancer and optimization strategies to obtain a better diffracting crystal. The best diffracting crystal was obtained in a condition containing 0.1 M Tris–HCl pH 7.4, 0.2 M MgCl 2 , 30% PEG 3350 using the hanging-drop vapour-diffusion method. A highly redundant in-house data set was collected to 2.75 Å resolution with 99% completeness. The presence of the mSox17-HMG–DNA complex within the crystals was confirmed and Matthews analysis indicated the presence of one complex per asymmetric unit

  1. Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, Chiho [Faculty of Pharmaceutical Sciences, Kyushu University (Japan); Quantum Beam Science Directorate, Japan Atomic Energy Agency (Japan); Taura, Futoshi [Faculty of Pharmaceutical Sciences, Kyushu University (Japan); Tamada, Taro; Shoyama, Yoshinari [Quantum Beam Science Directorate, Japan Atomic Energy Agency (Japan); Shoyama, Yukihiro; Tanaka, Hiroyuki [Faculty of Pharmaceutical Sciences, Kyushu University (Japan); Kuroki, Ryota [Quantum Beam Science Directorate, Japan Atomic Energy Agency (Japan); Morimoto, Satoshi [Faculty of Pharmaceutical Sciences, Kyushu University (Japan)

    2008-03-01

    Polyketide synthase-1 from C. sativa has been crystallized. The crystal diffracted to 1.55 Å resolution with sufficient quality for further structure determination. Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa (Mexican strain). PKS-1 was overproduced in Escherichia coli, purified and finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w/v) polyethylene glycol 3350 diffracted to 1.65 Å resolution and belonged to space group P1, with unit-cell parameters a = 54.3, b = 59.3, c = 62.6 Å, α = 69, β = 81, γ = 80°. Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w/v) polyethylene glycol 3350 diffracted to 1.55 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 54.3, b = 110, c = 130 Å. These data will enable us to determine the crystal structure of PKS-1.

  2. Crystallization and preliminary X-ray diffraction studies of polyketide synthase-1 (PKS-1) from Cannabis sativa

    International Nuclear Information System (INIS)

    Taguchi, Chiho; Taura, Futoshi; Tamada, Taro; Shoyama, Yoshinari; Shoyama, Yukihiro; Tanaka, Hiroyuki; Kuroki, Ryota; Morimoto, Satoshi

    2008-01-01

    Polyketide synthase-1 from C. sativa has been crystallized. The crystal diffracted to 1.55 Å resolution with sufficient quality for further structure determination. Polyketide synthase-1 (PKS-1) is a novel type III polyketide synthase that catalyzes the biosynthesis of hexanoyl triacetic acid lactone in Cannabis sativa (Mexican strain). PKS-1 was overproduced in Escherichia coli, purified and finally crystallized in two different space groups. The crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M calcium acetate and 20%(w/v) polyethylene glycol 3350 diffracted to 1.65 Å resolution and belonged to space group P1, with unit-cell parameters a = 54.3, b = 59.3, c = 62.6 Å, α = 69, β = 81, γ = 80°. Another crystal obtained in 0.1 M HEPES buffer pH 7.5 containing 0.2 M sodium chloride and 20%(w/v) polyethylene glycol 3350 diffracted to 1.55 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 54.3, b = 110, c = 130 Å. These data will enable us to determine the crystal structure of PKS-1

  3. Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Gαi1

    International Nuclear Information System (INIS)

    Morikawa, Tomohito; Muroya, Ayumu; Nakajima, Yoshitaka; Tanaka, Takeshi; Hirai, Keiko; Sugio, Shigetoshi; Wakamatsu, Kaori; Kohno, Toshiyuki

    2007-01-01

    The K349P mutant of Gα i1 , which is unable to couple to G-protein-coupled receptors, has been crystallized and analyzed. The same crystallization conditions were applicable irrespective of the identity of the bound nucleotide or of the presence of the mutation. In order to understand the molecular mechanisms by which G-protein-coupled receptors (GPCRs) activate G proteins, the K349P mutant of Gα i1 (K349P), which is unable to couple to the muscarinic acetylcholine receptor, was prepared and its crystals were grown along with those of wild-type Gα i1 protein (WT). The two proteins were crystallized under almost identical conditions, thus enabling a detailed structural comparison. The crystallization conditions performed well irrespective of the identity of the bound nucleotide (GDP or GTPγS) and the crystals diffracted to resolutions of 2.2 Å (WT·GDP), 2.8 Å (WT·GTPγS), 2.6 Å (K349P·GDP) and 3.2 Å (K349P·GTPγS)

  4. Crystallization and preliminary X-ray diffraction analysis of protein 14 from Sulfolobus islandicus filamentous virus (SIFV)

    International Nuclear Information System (INIS)

    Goulet, Adeline; Spinelli, Silvia; Campanacci, Valérie; Porciero, Sophie; Blangy, Stéphanie; Garrett, Roger A.; Tilbeurgh, Herman van; Leulliot, Nicolas; Basta, Tamara; Prangishvili, David; Cambillau, Christian

    2006-01-01

    Crystals of S. islandicus filamentous virus (SIFV) protein 14 have been grown at 293 K. Crystals belong to space group P6 2 22 or P6 4 22 and diffract to a resolution of 2.95 Å. A large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. Here, the crystallization of protein 14 from the archaeal virus SIFV is reported. This protein, which contains 111 residues (MW 13 465 Da), was cloned and expressed in Escherichia coli with an N-terminal His 6 tag and purified to homogeneity. The tag was subsequently cleaved and the protein was crystallized using PEG 1000 or PEG 4000 as a precipitant. Large crystals were obtained of the native and the selenomethionine-labelled protein using sitting drops of 100–300 nl. Crystals belong to space group P6 2 22 or P6 4 22, with unit-cell parameters a = b = 68.1, c = 132.4 Å. Diffraction data were collected to a maximum acceptable resolution of 2.95 and 3.20 Å for the SeMet-labelled and native protein, respectively

  5. Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

    Science.gov (United States)

    Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak; Escalante, Carlos R

    2015-04-01

    The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

  6. Purification, crystallization and preliminary X-ray analysis of a Nup107–Nup133 heterodimeric nucleoporin complex

    International Nuclear Information System (INIS)

    Boehmer, Thomas; Schwartz, Thomas U.

    2007-01-01

    A heterodimeric complex consisting of the C-terminal domains of human nucleoporins Nup107 and Nup133 was purified and crystallized and complete diffraction data sets were collected. The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and Nup133 are members of the essential Nup107–160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-terminal domains of human Nup107 and Nup133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18–22% PEG 3350 belong to space group P2 1 2 1 2 1 and diffracted to 2.9 Å. Native and seleno-l-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 Å, respectively. Structure determination of this complex will give the first insights into the protein–protein interactions within a core module of the NPC

  7. Purification, crystallization and preliminary X-ray analysis of a Nup107–Nup133 heterodimeric nucleoporin complex

    Energy Technology Data Exchange (ETDEWEB)

    Boehmer, Thomas; Schwartz, Thomas U., E-mail: tus@mit.edu [Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 (United States)

    2007-09-01

    A heterodimeric complex consisting of the C-terminal domains of human nucleoporins Nup107 and Nup133 was purified and crystallized and complete diffraction data sets were collected. The nuclear pore complex (NPC), the sole gateway of traffic between the nucleus and the cytoplasm, is built up from multiple copies of about 30 proteins collectively termed nucleoporins (nups). Nups are organized into distinct subcomplexes. Nup107 and Nup133 are members of the essential Nup107–160 subcomplex, a component of the central NPC architecture. A dimeric complex of the C-terminal domains of human Nup107 and Nup133 was expressed from a bicistronic vector in Escherichia coli, purified and crystallized in two different crystal forms. Crystals grown in the presence of 18–22% PEG 3350 belong to space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 2.9 Å. Native and seleno-l-methionine-derivative crystals grown in the presence of 1.1 M sodium malonate belong to space group C2 and diffracted to 2.55 and 2.9 Å, respectively. Structure determination of this complex will give the first insights into the protein–protein interactions within a core module of the NPC.

  8. Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

    International Nuclear Information System (INIS)

    Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

    2009-01-01

    A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

  9. Expression, crystallization and preliminary X-ray analysis of the periplasmic stress sensory protein RseB from Escherichia coli

    International Nuclear Information System (INIS)

    Wollmann, Petra; Zeth, Kornelius

    2006-01-01

    The periplasmic stress protein RseB from E. coli was cloned, expressed and crystallized. Crystallographic data are presented and structure solution using the multiple isomorphous replacement approach (MIR) is in progress. Sensing external stress in the bacterial periplasm and signal transduction to the cytoplasm are important functions of the CpxAR, Bae and σ E signalling pathways. In Escherichia coli, the σ E pathway can be activated through degradation of the antisigma factor RseA by DegS and YaeL. The periplasmic protein RseB plays an important role in this pathway by exerting a direct or indirect negative effect on YaeL cleavage efficiency. RseB from E. coli, missing the periplasmic signal sequence (RseB ΔN ), was cloned, expressed, purified and crystallized. Crystals were obtained in two different forms belonging to space group P42 1 2 (form I) and C222 1 (form II) and diffracted to 2.8 and 2.4 Å resolution, respectively. In crystal form I two copies of the protein were located in the asymmetric unit according to heavy-atom analysis, while crystal form II contained three copies

  10. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    International Nuclear Information System (INIS)

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-01-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

  11. Crystallization and preliminary X-ray characterization of Epstein–Barr virus BHRF1 in complex with a benzoylurea peptidomimetic

    International Nuclear Information System (INIS)

    Caria, Sofia; Chugh, Srishti; Nhu, Duong; Lessene, Guillaume; Kvansakul, Marc

    2012-01-01

    The expression and purification of Epstein–Barr virus BHRF1 as well as its co-crystallization with a peptidomimetic are described. BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein–Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains. Screening of a library of peptidomimetic compounds based on the benzoylurea scaffold that mimics critical Bim BH3 domain side chains against BHRF1 led to the identification of an inhibitor of BHRF1 that displays micromolar affinity. Single crystals were obtained from the co-crystallization of recombinant BHRF1 protein with this peptidomimetic compound. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 66.8, b = 91.1, c = 151.9 Å. Diffraction data were collected to 2.11 Å resolution on the MX2 beamline at the Australian Synchrotron

  12. Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

    International Nuclear Information System (INIS)

    Rocha, Rita; Barbosa Pereira, Pedro José; Santos, Manuel A. S.; Macedo-Ribeiro, Sandra

    2010-01-01

    The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6 1 22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

  13. Crystallization and preliminary X-ray analysis of RsbS from Moorella thermoacetica at 2.5 Å resolution

    International Nuclear Information System (INIS)

    Quin, Maureen; Newman, Joseph; Firbank, Susan; Lewis, Richard J.; Marles-Wright, Jon

    2008-01-01

    Crystallization and selenium substructure solution of RsbS from Moorella thermoacetica, the first ab initio phased crystal structure from Diamond. The thermophilic bacterium Moorella thermoacetica possesses an rsb operon that is related to the genetic locus common to many Gram-positive bacteria that regulates the activity of the stress-responsive sigma factor σ B . One of the gene products of this operon is RsbS, a single STAS-domain protein that is a component of higher order assemblies in Bacillus subtilis known as ‘stressosomes’. It is expected that similar complexes are found in M. thermoacetica, but in this instance regulating the biosynthesis of cyclic di-GMP, a ubiquitous secondary messenger. Selenomethionine-labelled MtRsbS protein was crystallized at room temperature using the hanging-drop vapour-diffusion method. Crystals belonging to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.07, b = 60.52, c = 89.28 Å, diffracted to 2.5 Å resolution on beamline I04 of the Diamond Light Source. The selenium substructure was solved using SHELX and it is believed that this represents the first reported ab initio crystal structure to be solved using diffraction data collected at DLS

  14. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Bin, E-mail: ren@csb.ki.se [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden); Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu [Université Paris-Sud, Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8621, F-91405 Orsay CEDEX (France); Ladenstein, Rudolf [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden)

    2007-05-01

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C222{sub 1} with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution.

  15. Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

    International Nuclear Information System (INIS)

    Ullah, Anwar; Giuseppe, Priscila Oliveira de; Murakami, Mario Tyago; Trevisan-Silva, Dilza; Wille, Ana Carolina Martins; Chaves-Moreira, Daniele; Gremski, Luiza Helena; Silveira, Rafael Bertoni da; Sennf-Ribeiro, Andrea; Chaim, Olga Meiri; Veiga, Silvio Sanches; Arni, Raghuvir Krishnaswamy

    2011-01-01

    Wild-type and H12A-mutant class II phospholipase D from L. intermedia venom were crystallized; the crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively. Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12 1 1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5 Å, β = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60 Å, respectively

  16. Crystallization and preliminary X-ray diffraction analysis of the peripheral light-harvesting complex LH2 from Marichromatium purpuratum.

    Science.gov (United States)

    Cranston, Laura J; Roszak, Aleksander W; Cogdell, Richard J

    2014-06-01

    LH2 from the purple photosynthetic bacterium Marichromatium (formerly known as Chromatium) purpuratum is an integral membrane pigment-protein complex that is involved in harvesting light energy and transferring it to the LH1-RC `core' complex. The purified LH2 complex was crystallized using the sitting-drop vapour-diffusion method at 294 K. The crystals diffracted to a resolution of 6 Å using synchrotron radiation and belonged to the tetragonal space group I4, with unit-cell parameters a=b=109.36, c=80.45 Å. The data appeared to be twinned, producing apparent diffraction symmetry I422. The tetragonal symmetry of the unit cell and diffraction for the crystals of the LH2 complex from this species reveal that this complex is an octamer.

  17. Expression, purification, crystallization and preliminary X-ray studies of the Ebola VP35 interferon inhibitory domain

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Ginder, Nathaniel D.; Nix, Jay C.; Basler, Christopher F.; Honzatko, Richard B.; Amarasinghe, Gaya K.

    2009-01-01

    Native and selenomethionine-labeled crystals of Ebola VP35 interferon inhibitory domain were obtained by the hanging-drop vapor-diffusion method. Ebola VP35 is a multifunctional protein that is important for host immune suppression and pathogenesis. VP35 contains an N-terminal oligomerization domain and a C-terminal interferon inhibitory domain (IID). Mutations within the VP35 IID result in loss of host immune suppression. Here, efforts to crystallize recombinantly overexpressed VP35 IID that was purified from Escherichia coli are described. Native and selenomethionine-labeled crystals belonging to the orthorhombic space group P2 1 2 1 2 1 were obtained by the hanging-drop vapor-diffusion method and diffraction data were collected at the ALS synchrotron

  18. Purification, crystallization and preliminary X-ray diffraction studies of D-tagatose 3-epimerase from Pseudomonas cichorii.

    Science.gov (United States)

    Yoshida, Hiromi; Yamada, Mitsugu; Nishitani, Takeyori; Takada, Goro; Izumori, Ken; Kamitori, Shigehiro

    2007-02-01

    D-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of D-psicose has not been reported with epimerases other than P. cichorii D-TE and D-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 A, beta = 102.82 degrees . Diffraction data were collected to 2.5 A resolution. The asymmetric unit is expected to contain four molecules.

  19. Purification, crystallization and preliminary X-ray crystallographic analysis of human ppGpp hydrolase, Mesh1

    Directory of Open Access Journals (Sweden)

    Dawei Sun

    2010-09-01

    Full Text Available Bacterial SpoT is a Mn2+-dependent pyrophosphohydrolase to hydrolyze guanosine 3’-diphosphate-5’-diphosphate to guanosine diphosphate and pyrophosphate. In this study, SpoT ortholog from Homo sapiens (hMesh1, was over-expressed in Escherichia coli, purified and crystallized using hanging-drop vapour-diffusion method with polyethylene glycol and sodium citrate. The native crystal of hMesh1 was diffracted to 2.1 Å using a synchrotron-radiation source and belonged to the monoclinic space group P21 with cell dimensions of a = 53.27 Å, b = 62.61 Å, c = 52.45 Å and β= 94.96˚. The crystal contains two molecules in the asymmetric unit, with a solvent content of 44% and a Matthews coefficient VM value of 2.18 Å3/Da.

  20. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2

    International Nuclear Information System (INIS)

    Cheng, Xia; Lu, Guangwen; Qi, Jianxun; Cheng, Hao; Gao, Feng; Wang, Jundong; Yan, Jinghua

    2010-01-01

    Crystals of SAICAR synthase from S. suis serotype 2 were obtained in the presence of 40 mM aspartic acid substrate; they belonged to space group P2 and diffracted to 2.8 Å resolution. Phosphoribosylaminoimidazole-succinocarboxamide synthase (SAICAR synthase) plays an essential role in the de novo biosynthesis of purine nucleotides. In this study, the SAICAR synthase from Streptococcus suis was cloned and overexpressed in Escherichia coli. The subsequent product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.8 Å resolution and belonged to space group P2, with unit-cell parameters a = 70.2, b = 52.2, c = 153.9 Å, β = 102.8°

  1. The macromolecular complex of ICP and falcipain-2 from Plasmodium: preparation, crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Hansen, Guido; Schwarzloh, Britta; Rennenberg, Annika; Heussler, Volker T.; Hilgenfeld, Rolf

    2011-01-01

    The macromolecular complex of ICP (inhibitor of cysteine proteases) from P. berghei and falcipain-2 from P. falciparum has been prepared and crystallized, and a diffraction data set has been collected to a resolution of 2.6 Å. The malaria parasite Plasmodium depends on the tight control of cysteine-protease activity throughout its life cycle. Recently, the characterization of a new class of potent inhibitors of cysteine proteases (ICPs) secreted by Plasmodium has been reported. Here, the recombinant production, purification and crystallization of the inhibitory C-terminal domain of ICP from P. berghei in complex with the P. falciparum haemoglobinase falcipain-2 is described. The 1:1 complex was crystallized in space group P4 3 , with unit-cell parameters a = b = 71.15, c = 120.09 Å. A complete diffraction data set was collected to a resolution of 2.6 Å

  2. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    International Nuclear Information System (INIS)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He; Su, Xiao-Dong

    2006-01-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni 2+ -chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2 1 , with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°

  3. Preparation, crystallization and preliminary X-ray analysis of the methionine synthase (MetE) from Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Tian-Min; Zhang, Xiao-Yan; Li, Lan-Fen; Liang, Yu-He, E-mail: liangyh@pku.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871 (China); Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing 100871 (China)

    2006-10-01

    Methionine synthase (MetE) from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.2 Å resolution. The Streptococcus mutans metE gene encodes methionine synthase (MetE), which catalyzes the direct transfer of a methyl group from methyltetrahydrofolate to homocysteine in the last step of methionine synthesis. metE was cloned into pET28a and the gene product was expressed at high levels in the Escherichia coli strain BL21 (DE3). MetE was purified to homogeneity using Ni{sup 2+}-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.2 Å resolution. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 52.85, b = 99.48, c = 77.88 Å, β = 94.55°.

  4. Crystallization and preliminary X-ray characterization of a PaaX-like protein from Sulfolobus solfataricus P2

    International Nuclear Information System (INIS)

    Cao, Yi; Lou, Zhiyong; Sun, Yuna; Xue, Fei; Feng, Changzeng; Gong, Xiaocui; Yang, Dongmei; Bartlam, Mark; Meng, Zhaohui; Zhang, Keqin

    2009-01-01

    In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. PaaX is a global regulator of the phenylacetyl-coenzyme A catabolon that adjusts the expression of different operons to that of the paa-encoded central pathway. In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Diffraction data were obtained to a resolution of 3.0 Å using synchrotron radiation at the Photon Factory. The crystal belonged to space group P321, with unit-cell parameters a = 86.4, b = 86.4, c = 105.5 Å

  5. Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the SH3 domain of human AHI1

    International Nuclear Information System (INIS)

    Shi, Zhuliang; Liang, Ning; Xu, Wei; Li, Kuai; Sheng, Guoqing; Liu, Jinsong; Xu, Aimin; Li, Xiao-Jiang; Wu, Donghai

    2009-01-01

    The SH3 domain of human AHI1 has been cloned and expressed in Escherichia coli. The protein was purified by affinity and size-exclusion chromatography and was crystallized using the sitting-drop vapour-diffusion method at 293 K. The SH3 domain of human AHI1 was cloned and expressed in Escherichia coli. The protein was purified by affinity and size-exclusion chromatography and was crystallized using the sitting-drop vapour-diffusion method at 293 K. A complete data set was collected to 2.5 Å resolution at 110 K. The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = 67.377, b = 67.377, c = 98.549 Å

  6. Crystallization and preliminary X-ray analysis of the HA3 component of Clostridium botulinum type C progenitor toxin

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Toshio; Tonozuka, Takashi; Kotani, Mao; Obata, Kanae [Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); Oguma, Keiji [Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558 (Japan); Nishikawa, Atsushi, E-mail: nishikaw@cc.tuat.ac.jp [Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); CREST, Japan Science and Technology Agency (Japan)

    2007-12-01

    HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. HA3, a 70 kDa haemagglutinating protein, is a precursor form of HA3a and HA3b, the subcomponents of Clostridium botulinum type C 16S progenitor toxin. In this report, recombinant HA3 protein was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.6 Å resolution and the crystal belonged to the hexagonal space group P6{sub 3}. Matthews coefficient and self-rotation function calculations indicate that there is probably one molecule of HA3 in the asymmetric unit. A search for heavy-atom derivatives has been undertaken.

  7. Crystallization, X-ray diffraction analysis and preliminary structure determination of the polygalacturonase PehA from Agrobacterium vitis

    International Nuclear Information System (INIS)

    Vordtriede, Paul B.; Yoder, Marilyn D.

    2008-01-01

    The acidic polygalacturonase PehA from A. vitis has been crystallized. A molecular-replacement solution indicated a right-handed parallel β-helix fold. Polygalacturonases are pectate-degrading enzymes that belong to glycoside hydrolase family 28 and hydrolyze the α-1,4 glycosidic bond between neighboring galacturonasyl residues of the homogalacturonan substrate. The acidic polygalacturonase PehA from Agrobacterium vitis was overexpressed in Escherichia coli, where it accumulated in the periplasmic fraction. It was purified to homogeneity via a two-step chromatography procedure and crystallized using the hanging-drop vapour-diffusion technique. PehA crystals belonged to space group P2 1 , with unit-cell parameters a = 52.387, b = 62.738, c = 149.165 Å, β = 89.98°. Crystals diffracted to 1.59 Å resolution and contained two molecules per asymmetric unit. An initial structure determination by molecular replacement indicated a right-handed parallel β-helix fold

  8. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-like protein

    NARCIS (Netherlands)

    Luberacki, B.; Weyand, M.; Seitz, H.U.; Koch, W.; Oecking, C.; Ottmann, C.

    2008-01-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 angstrom resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled

  9. Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395

    International Nuclear Information System (INIS)

    Biswas, Maitree; Khamrui, Susmita; Sen, Udayaditya; Dasgupta, Jhimli

    2011-01-01

    The chemotaxis response regulator CheY4 from V. cholerae has been cloned, overexpressed, purified and crystallized in monoclinic and hexagonal space groups; the crystals diffracted to 1.67 and 1.9 Å resolution, respectively. Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1–CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni–NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, β = 96.5°, whereas crystals grown in space group P3 2 21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°

  10. Purification, crystallization and preliminary X-ray crystallographic analysis of a methanol dehydrogenase from the marine bacterium Methylophaga aminisulfidivorans MPT

    International Nuclear Information System (INIS)

    Choi, Jin Myung; Kim, Hee Gon; Kim, Jeong-Sun; Youn, Hyung-Seop; Eom, Soo Hyun; Yu, Sung-Lim; Kim, Si Wouk; Lee, Sung Haeng

    2011-01-01

    In order to obtain molecular insights into the methanol-oxidizing system of M. aminisulfidivorans, a native heterotetrameric α 2 β 2 methanol dehydrogenase complex was directly purified from M. aminisulfidivorans MP T grown in the presence of methanol and crystallized. Methylophaga aminisulfidivorans MP T is a marine methylotrophic bacterium that utilizes C 1 compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c L . In order to obtain molecular insights into the oxidation mechanism, a native heterotetrameric α 2 β 2 MDH complex was directly purified from M. aminisulfidivorans MP T grown in the presence of methanol and crystallized. The crystal diffracted to 1.7 Å resolution and belonged to the monoclinic space group P2 1 (unit-cell parameters a = 63.9, b = 109.5, c = 95.6 Å, β = 100.5°). The asymmetric unit of the crystal contained one heterotetrameric complex, with a calculated Matthews coefficient of 2.24 Å 3 Da −1 and a solvent content of 45.0%

  11. Crystallization and preliminary X-ray analysis of the N-terminal domain of human thioredoxin-interacting protein

    International Nuclear Information System (INIS)

    Polekhina, Galina; Ascher, David Benjamin; Kok, Shie Foong; Waltham, Mark

    2011-01-01

    The N-terminal domain of thioredoxin-interacting protein has been expressed, purified and crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation. Thioredoxin-interacting protein (TXNIP) is a negative regulator of thioredoxin and its roles in the pathologies of diabetes and cardiovascular diseases have marked it out as a potential drug target. Expression of TXNIP is robustly induced under various stress conditions such as high glucose, heat shock, UV, H 2 O 2 and mechanical stress amongst others. Elevated levels of TXNIP result in the sequestration and inactivation of thioredoxin, leading to cellular oxidative stress. For some time, this was the only known function of TXNIP; however, more recently the protein has been shown to play a role in regulation of glucose uptake and activation of the inflammasome. Based on the primary sequence, TXNIP is remotely related to β-arrestins, which include the visual arrestins. TXNIP has thus been classified as a member of the α-arrestin family, which to date includes five other members. None of the other α-arrestins are known to interact with thioredoxin, although curiously one has been implicated in glucose uptake. In order to gain insight into the structure–function relationships of the α-arrestin protein family, and particularly that of TXNIP, the N-terminal domain of TXNIP has been crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation

  12. Crystallization and preliminary X-ray crystallographic studies of Mycobacterium tuberculosis CRP/FNR family transcription regulator

    International Nuclear Information System (INIS)

    Akif, Mohd; Akhter, Yusuf; Hasnain, Seyed E.; Mande, Shekhar C.

    2006-01-01

    The CRP/FNR family transcription factor from M. tuberculosis H37Rv has been crystallized in space group P2 1 2 1 2 1 in the absence of cAMP. The crystals show the presence of a dimeric molecule in the asymmetric unit. CRP/FNR family members are transcription factors that regulate the transcription of many genes in Escherichia coli and other organisms. Mycobacterium tuberculosis H37Rv contains a probable CRP/FNR homologue encoded by the open reading frame Rv3676. The deletion of this gene is known to cause growth defects in cell culture, in bone marrow-derived macrophages and in a mouse model of tuberculosis. The mycobacterial gene Rv3676 shares ∼32% sequence identity with prototype E. coli CRP. The structure of the protein might provide insight into transcriptional regulation in the pathogen by this protein. The M. tuberculosis CRP/FNR transcription regulator was crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 54.1, b = 84.6, c = 101.2 Å. The crystal diffracted to a resolution of 2.9 Å. Matthews coefficient and self-rotation function calculations reveal the presence of two monomers in the asymmetric unit

  13. Protein expression, crystallization and preliminary X-ray crystallographic studies of LidA from Legionella pneumophila

    International Nuclear Information System (INIS)

    Zhu, Wenzhuang; Meng, Geng; Liu, Yong; Zhang, Feiyun; Zheng, Xiaofeng

    2011-01-01

    The crystallization of recombinant lidA, a translocated substrate of the Legionella pneumophila Dot/Icm type IV secretion system, is reported. Crystals were obtained and diffracted to 2.75 Å in space group P2 1 2 1 2 1 . LidA, a translocated substrate of the Legionella pneumophila Dot/Icm type IV secretion system, is associated with maintenance of bacterial integrity and interferes with the early secretory pathway. However, the precise mechanism of LidA in these processes remains elusive. To further investigate the structure and function of LidA, the full-length protein was successfully expressed in Escherichia coli and purified. LidA was crystallized using sitting-drop vapour diffusion and diffracted to a resolution of 2.75 Å. The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 57.5, b = 64.5, c = 167.3 Å, α = β = γ = 90°. There is one molecule per asymmetric unit

  14. Crystallization and Preliminary X-ray Analysis of Bacteriophasge T4 UvsY Recombination Mediator Protein

    International Nuclear Information System (INIS)

    Xu, H.; Beernink, H.; Rould, M.; Morrical, S.

    2006-01-01

    Bacteriophage T4 UvsY protein is considered to be the prototype of recombination mediator proteins, a class of proteins which assist in the loading of recombinases onto DNA. Wild-type and Se-substituted UvsY protein have been expressed and purified and crystallized by hanging-drop vapor diffusion. The crystals diffract to 2.4 (angstrom) using in-house facilities and to 2.2 (angstrom) at NSLS, Brookhaven National Laboratory. The crystals belong to space group P422, P4 2 22, P42 1 2 or P4 2 2 1 2, the ambiguity arising from pseudo-centering, with unit-cell parameters a = b = 76.93, c = 269.8 (angstrom). Previous biophysical characterization of UvsY indicates that it exists primarily as a hexamer in solution. Along with the absence of a crystallographic threefold, this suggests that the asymmetric unit of these crystals is likely to contain either three monomers, giving a solvent content of 71%, or six monomers, giving a solvent content of 41%

  15. Crystallization and preliminary X-ray crystallographic analysis of two vascular apoptosis-inducing proteins (VAPs) from Crotalus atrox venom

    International Nuclear Information System (INIS)

    Igarashi, Tomoko; Oishi, Yuko; Araki, Satohiko; Mori, Hidezo; Takeda, Soichi

    2006-01-01

    Vascular apoptosis-inducing protein 1 (VAP1) and VAP2 from C. atrox venom were crystallized in variety of different crystal forms. Diffraction data sets were obtained to 2.5 and 2.15 Å resolution for VAP1 and VAP2, respectively. VAPs are haemorrhagic snake-venom toxins belonging to the reprolysin family of zinc metalloproteinases. In vitro, VAPs induce apoptosis specifically in cultured vascular endothelial cells. VAPs have a modular structure that bears structural homology to mammalian ADAMs (a disintegrin and metalloproteinases). VAP1 is a homodimer with a MW of 110 kDa in which the monomers are connected by a single disulfide bridge. VAP2 is homologous to VAP1 and exists as a monomer with a MW of 55 kDa. In the current study, several crystal forms of VAP1 and VAP2 were obtained using the vapour-diffusion method and diffraction data sets were collected using SPring-8 beamlines. The best crystals of VAP1 and VAP2 generated data sets to 2.5 and 2.15 Å resolution, respectively

  16. Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Molina, Rafael [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); González, Ana; Moscoso, Miriam; García, Pedro [Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Stelter, Meike; Kahn, Richard [Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF, Laboratoire de Cristallographie Macromoléculaire, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 1 (France); Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain)

    2007-09-01

    The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L{sub III} absorption edge using synchrotron radiation.

  17. Crystallization and preliminary X-ray crystallographic analysis of the ArsM arsenic(III) S-adenosylmethionine methyltransferase

    International Nuclear Information System (INIS)

    Marapakala, Kavitha; Ajees, A. Abdul; Qin, Jie; Sankaran, Banumathi; Rosen, Barry P.

    2010-01-01

    A common biotransformation of arsenic is methylation to monomethylated, dimethylated and trimethylated species, which is catalyzed by the ArsM (or AS3MT) arsenic(III) S-adenosylmethionine methyltransferase. ArsM from the acidothermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized by the hanging-drop vapor-diffusion method and diffraction data were collected to 1.76 Å resolution. Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency’s Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III) S-adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 84.85, b = 46.89, c = 100.35 Å, β = 114.25° and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76 Å

  18. Crystallization and preliminary X-ray diffraction analysis of ybfF, a new esterase from Escherichia coli K12

    Energy Technology Data Exchange (ETDEWEB)

    Park, Suk-Youl; Lee, Sang-Hak; Lee, Jieun; Jung, Che-Hun; Kim, Jeong-Sun, E-mail: jsunkim@chonnam.ac.kr [Department of Chemistry and Institute of Basic Sciences, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2007-12-01

    The crystallization of ybfF, a new esterase from E. coli, and the collection of diffraction data to 1.1 Å resolution are reported. The product of the recently discovered ybfF gene, which belongs to the esterase family, does not show high sequence similarity to other esterases. To provide the molecular background to the enzymatic mechanism of the ybfF esterase, the ybfF protein from Escherichia coli K12 (Ec-ybfF) was cloned, expressed and purified. The Ec-ybfF protein was crystallized from 60% Tacsimate and 0.1 M bis-Tris propane buffer pH 7.0. Diffraction data were collected to 1.10 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.09, b = 90.71, c = 92.88 Å. With two Ec-ybfF molecules in the asymmetric unit, the crystal volume per unit protein weight is 2.17 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 42%.

  19. Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Molina, Rafael; González, Ana; Moscoso, Miriam; García, Pedro; Stelter, Meike; Kahn, Richard; Hermoso, Juan A.

    2007-01-01

    The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2 1 2 1 2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L III absorption edge using synchrotron radiation

  20. Purification, crystallization and preliminary X-ray diffraction analysis of disease-related mutants of p97

    International Nuclear Information System (INIS)

    Tang, Wai-Kwan; Li, Dongyang; Esser, Lothar; Xia, Di

    2009-01-01

    Mutations in the human AAA+ protein p97 cause a disease in humans called IBMPFD. How these mutations affect the structure and function of p97 is unknown. Here, the crystallization of three disease-related mutants of p97 in the presence of ATPγS are reported. The human type II AAA+ protein p97 participates in various cellular activities, presumably through its involvement in the ubiquitin–proteasome degradation pathway. Mutations in p97 have been implicated in patients with inclusion-body myopathy associated with Paget’s disease of the bone and frontotemporal dementia (IBMPFD). In this work, three mutant p97 N-D1 fragments, R86A, R95G and R155H, were crystallized in the presence of ATPγS with PEG 3350 as a main precipitant, yielding two different crystal forms. The R155H mutant crystal belonged to space group R3, with unit-cell parameters in the hexagonal setting of a = b = 134.2, c = 182.9 Å, and was merohedrally twinned, with an estimated twin fraction of 0.34. The crystals of the R86A and R95G mutants belonged to space group P1, with similar unit-cell parameters of a = 90.89, b = 102.6, c = 107.2 Å, α = 97.5, β = 90.6, γ = 91.5° and a = 92.76, b = 103.7, c = 107.7 Å, α = 97.7, β = 91.9, γ = 89.7°, respectively

  1. Crystallization and preliminary X-ray crystallographic analysis of enoyl-ACP reductase III (FabL) from Bacillus subtilis

    International Nuclear Information System (INIS)

    Kim, Kook-Han; Park, Joon Kyu; Ha, Byung Hak; Moon, Jin Ho; Kim, Eunice EunKyeong

    2007-01-01

    Enoyl-ACP reductase III (FabL) from B. subtilis has been overexpressed, purified and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120°, and data were collected to 2.5 Å resolution using synchrotron radiation. Enoyl-[acyl-carrier protein] reductase (enoyl-ACP reductase; ENR) is a key enzyme in type II fatty-acid synthase that catalyzes the last step in each elongation cycle. It has been considered as an antibiotic target since it is an essential enzyme in bacteria. However, recent studies indicate that some pathogens have more than one ENR. Bacillus subtilis is reported to have two ENRs, namely BsFabI and BsFabL. While BsFabI is similar to other FabIs, BsFabL shows very little sequence similarity and is NADPH-dependent instead of NADH-dependent as in the case of FabI. In order to understand these differences on a structural basis, BsFabL has been cloned, expressed and and crystallized. The crystal belongs to space group P622, with unit-cell parameters a = b = 139.56, c = 62.75 Å, α = β = 90, γ = 120° and one molecule of FabL in the asymmetric unit. Data were collected using synchrotron radiation (beamline 4A at the Pohang Light Source, Korea). The crystal diffracted to 2.5 Å resolution

  2. Crystallization and preliminary X-ray structural studies of hemoglobin A2 and hemoglobin E, isolated from the blood samples of β-thalassemic patients

    International Nuclear Information System (INIS)

    Dasgupta, Jhimli; Sen, Udayaditya; Choudhury, Debi; Datta, Poppy; Chakrabarti, Abhijit; Chakrabarty, Sudipa Basu; Chakrabarty, Amit; Dattagupta, J.K.

    2003-01-01

    Hemoglobin A 2 (α 2 δ 2 ), a minor (2-3%) component of circulating red blood cells, acts as an anti-sickling agent and its elevated concentration in β-thalassemia is a useful clinical diagnostic. In β-thalassemia major, where there is a failure of β-chain production, HbA 2 acts as the predominant oxygen deliverer. Hemoglobin E, is another common abnormal hemoglobin, caused by splice site mutation in exon 1 of β globin gene, when combines with β-thalassemia, causes severe microcytic anemia. The purification, crystallization, and preliminary structural studies of HbA 2 and HbE are reported here. HbA 2 and HbE are purified by cation exchange column chromatography in presence of KCN from the blood samples of individuals suffering from β-thalassemia minor and Eβ-thalassemia. X-ray diffraction data of HbA 2 and HbE were collected upto 2.1 and 1.73 A, respectively. HbA 2 crystallized in space group P2 1 with unit cell parameters a=54.33 A, b=83.73 A, c=62.87 A, and β=99.80 degree sign whereas HbE crystallized in space group P2 1 2 1 2 1 with unit cell parameters a=60.89 A, b=95.81 A, and c=99.08 A. Asymmetric unit in each case contains one Hb tetramer in R 2 state

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    Energy Technology Data Exchange (ETDEWEB)

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J., E-mail: s.j.matthews@imperial.ac.uk [Imperial College London, South Kensington, London SW7 2AZ (United Kingdom)

    2015-05-20

    In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it

  4. Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB

    International Nuclear Information System (INIS)

    Garnett, James A.; Diallo, Mamou; Matthews, Steve J.

    2015-01-01

    In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher pathway that plays a major role in both early biofilm formation and host-cell adhesion. Initial attempts at crystallizing the chaperone EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. This is the first time that this refolding strategy has been used to purify CU chaperones. Pili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone–usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be

  5. Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase from porcine kidney in complex with nicotinate mononucleotide

    International Nuclear Information System (INIS)

    Youn, Hyung-Seop; Kim, Mun-Kyoung; Kang, Gil Bu; Kim, Tae Gyun; An, Jun Yop; Lee, Jung-Gyu; Park, Kyoung Ryoung; Lee, Youngjin; Fukuoka, Shin-Ichi; Eom, Soo Hyun

    2012-01-01

    Crystals of S. scrofa quinolinate phosphoribosyltransferase purified from porcine kidney in complex with nicotinate mononucleotidewere obtained and diffraction data were collected and processed to 2.1 Å resolution. Quinolinate phosphoribosyltransferase (QAPRTase) is a key enzyme in NAD biosynthesis; it catalyzes the formation of nicotinate mononucleotide (NAMN) from quinolinate and 5-phosphoribosyl-1-pyrophosphate. In order to elucidate the mechanism of NAMN biosynthesis, crystals of Sus scrofa QAPRTase (Ss-QAPRTase) purified from porcine kidney in complex with NAMN were obtained and diffraction data were collected and processed to 2.1 Å resolution. The Ss-QAPRTase–NAMN cocrystals belonged to space group P321, with unit-cell parameters a = 119.1, b = 119.1, c = 93.7 Å, γ = 120.0°. The Matthews coefficient and the solvent content were estimated as 3.10 Å 3 Da –1 and 60.3%, respectively, assuming the presence of two molecules in the asymmetric unit

  6. Expression, purification, crystallization and preliminary X-ray diffraction analysis of grass carp β{sub 2}-microglobulin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weihong [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Chu, Fuliang [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Peng, Hao [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Zhang, Jianhua [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Qi, Jianxun; Jiang, Fan [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Xia, Chun, E-mail: xiachun@cau.edu.cn [Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China); Gao, Feng, E-mail: xiachun@cau.edu.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100080 (China); Department of Microbiology and Immunology, College of Veterinary Medicine, China Agricultural University, Beijing 100094 (China)

    2008-03-01

    Grass carp β{sub 2}-microglobulin was expressed in E. coli, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method with PEG 2K as precipitant. The crystals obtained belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. β{sub 2}-Microglobulin (β{sub 2}m) is an essential subunit of MHC I molecules; it stabilizes the structure of MHC I and plays a pivotal role in coreceptor recognition. To date, structures of β{sub 2}m have been solved for three different mammals: human, mouse and cattle. In order to illuminate the molecular evolutionary origin of β2m, an understanding of its structure in lower vertebrates becomes important. Here, grass carp (Ctenopharyngodon idellus) β{sub 2}m (Ctid-β{sub 2}m) was expressed, purified and crystallized. Diffraction data were collected to a resolution of 2.5 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.72, b = 40.65, c = 71.12 Å. The Matthews coefficient and the solvent content were calculated to be 2.56 Å Da{sup −1} and 52.07%, respectively, for one molecule per asymmetric unit. The structure has been solved by molecular replacement using monomeric human β{sub 2}m as a model.

  7. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    International Nuclear Information System (INIS)

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2006-01-01

    The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

  8. Crystallization and preliminary X-ray diffraction studies of the abscisic acid receptor PYL3 and its complex with pyrabactin

    International Nuclear Information System (INIS)

    Zhang, Xingliang; Wu, Wei; Chen, Zhongzhou

    2012-01-01

    Crystals of the abscisic acid receptor PYL3 and of the PYL3–pyrabactin complex were obtained and optimized in order to obtain high-quality diffraction data. Diffraction data sets were collected and processed to 2.5 and 1.83 Å resolution, respectively. Abscisic acid (ABA) modulates many developmental processes and responses to environmental stress. Recently, a family of pyrabactin resistance-like proteins (PYLs) in Arabidopsis thaliana were identified to be abscisic acid receptors. Although the 14 PYLs members share a similar sequence identity, they exhibit different responses toward pyrabactin. Apo-PYL3 is a dimer; however, its oligomeric state changes greatly on the addition of pyrabactin. Moreover, pyrabactin binds dimeric PYL3 in a nonproductive mode which prevents receptor activation and inhibition of PP2Cs. Here, the expression, purification and crystallization of apo-PYL3 and of PYL3 complexed with pyrabactin are reported. Diffraction data were optimized to 2.5 Å resolution for apo-PYL3 and to 1.83 Å resolution for PYL3–pyrabactin. The crystals of apo-PYL3 and PYL3–pyrabactin belonged to space groups P4 1 2 1 2 and P2 1 2 1 2 1 , respectively

  9. Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

    Science.gov (United States)

    Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

    2015-06-01

    Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

  10. Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix

    International Nuclear Information System (INIS)

    Förster, Charlotte; Perbandt, Markus; Brauer, Arnd B. E.; Brode, Svenja; Fürste, Jens P.; Betzel, Christian; Erdmann, Volker A.

    2006-01-01

    In order to investigate the identity elements of the E. coli tRNA Gly /GlyRS class II system, a tRNA Gly acceptor-stem microhelix was crystallized and a data set was collected to 2.0 Å resolution using synchrotron radiation. The tRNA Gly and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNA Gly . To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNA Gly acceptor-stem helix was crystallized. Data were collected to 2.0 Å resolution using synchrotron radiation. Crystals belong to space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 35.35, c = 130.82 Å, α = β = 90, γ = 120° and two molecules in the asymmetric unit

  11. Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxine

    International Nuclear Information System (INIS)

    Kobayashi, Jun; Yoshida, Hiromi; Chu, Huy Nhat; Yoshikane, Yu; Kamitori, Shigehiro; Yagi, Toshiharu

    2009-01-01

    Recombinant α-(N-acetylaminomethylene)succinic acid amidohydrolase from M. loti MAFF303099 was crystallized and diffraction data were collected at 2.7 Å resolution. α-(N-Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B 6 and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9 Å, β = 103.4°, and diffraction data were collected to 2.7 Å resolution. The V M value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit

  12. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

    Energy Technology Data Exchange (ETDEWEB)

    Vivan, Ana Luiza [Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS (Brazil); Dias, Márcio Vinícius Bertacini [Programa de Pós Graduação em Biofísica Molecular, Departamento de Física, IBILCE/UNESP, São José do Rio Preto, SP, 15054-000 (Brazil); Schneider, Cristopher Z. [Programa de Pós Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS (Brazil); Azevedo, Walter Filgueira Jr de; Basso, Luiz Augusto, E-mail: luiz.basso@pucrs.br; Santos, Diógenes Santiago, E-mail: luiz.basso@pucrs.br [Centro de Pesquisa em Biologia Molecular e Funcional, Instituto de Pesquisas Biomédicas, Pontifícia Universidade Católica do Rio Grande do Sul-PUCRS, Av. Ipiranga, 6681 Prédio 92A-TECNOPUC-Partenon, CEP 90619-900, Porto Alegre, RS (Brazil); Programa de Pós Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)

    2006-04-01

    The M. tuberculosis prephenate dehydratase was cloned, expressed, purified, crystallized by the hanging-drop vapour-diffusion method, and a complete data set collected to 3.2 Å resolution using synchrotron radiation. These results should pave the way for the three-dimensional structure determination of the enzyme and provide a framework on which to base the rational design of chemotherapeutic agents to treat tuberculosis. Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 Å resolution using a synchrotron-radiation source.

  13. Expression, purification, crystallization and preliminary X-ray analysis of YaeQ (XAC2396) from Xanthomonas axonopodis pv. citri

    International Nuclear Information System (INIS)

    Guzzo, Cristiane R.; Nagem, Ronaldo A. P.; Galvão-Botton, Leonor M. P.; Guimarães, Beatriz G.; Medrano, Francisco J.; Barbosa, João A. R. G.; Farah, Chuck S.

    2005-01-01

    The first crystallographic study of a member of the YaeQ family of proteins, which are conserved in a small group of Gram-negative bacteria, most of which are animal or plant pathogens, is reported. Diffraction data were collected to 1.9 Å resolution and an interpretable electron-density map was obtained. Xanthomonas axonopodis pv. citri YaeQ (XAC2396) is a member of a family of bacterial proteins conserved in several Gram-negative pathogens. Here, the cloning, expression, purification and crystallization of the 182-residue (20.6 kDa) YaeQ protein are described. Recombinant YaeQ containing selenomethionine was crystallized in space group P2 1 and crystals diffracted to 1.9 Å resolution at a synchrotron source. The unit-cell parameters are a = 39.75, b = 91.88, c = 48.03 Å, β = 108.37°. The calculated Matthews coefficient suggests the presence of two YaeQ molecules in the asymmetric unit. Initial experimental phases were calculated by the multiple-wavelength anomalous dispersion technique and an interpretable electron-density map was obtained

  14. Crystallization and preliminary X-ray analysis of the major peanut allergen Ara h 1 core region

    International Nuclear Information System (INIS)

    Cabanos, Cerrone; Urabe, Hiroyuki; Masuda, Taro; Tandang-Silvas, Mary Rose; Utsumi, Shigeru; Mikami, Bunzo; Maruyama, Nobuyuki

    2010-01-01

    In this study, the core region of Ara h 1, one of the major peanut allergens, has been overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.25 Å resolution. Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1 M sodium citrate pH 5.6, 0.1 M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 Å resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a = 156.521, b = 88.991, c = 158.971 Å, β = 107.144°. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan)

  15. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Inna; Kessler, Naama [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Moor, Nina [Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk (Russian Federation); Klipcan, Liron [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Koc, Emine [Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 (United States); Templeton, Paul [Department Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215 (United States); Spremulli, Linda [Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290 (United States); Safro, Mark, E-mail: mark.safro@weizmann.ac.il [Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  16. Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

    International Nuclear Information System (INIS)

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-01-01

    Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported. Clostridium perfringens is a ubiquitous environmental organism that is capable of causing a variety of diseases in mammals, including gas gangrene and necrotic enteritis in humans. The activity of a secreted hyaluronidase, attributed to the NagH protein, contributes to the pathogenicity of this organism. The family 84 catalytic module of one of the three homologues of NagH found in C. perfringens (ATCC 13124) has been cloned. The 69 kDa catalytic module of NagJ, here called GH84C, was overproduced in Escherichia coli and purified by immobilized metal-affinity chromatography (IMAC). Crystals belonging to space group I222 or I2 1 2 1 2 1 with unit-cell parameters a = 130.39, b = 150.05, c = 155.43 Å were obtained that diffracted to 2.1 Å. Selenomethionyl crystals have also been produced, leading to the possibility of solving the phase problem by MAD using synchrotron radiation

  17. Expression, purification, crystallization and preliminary X-ray diffraction analysis of rhesus macaque CD8αα homodimer

    International Nuclear Information System (INIS)

    Zong, Lili; Chen, Yong; Yan, Jinghua; Zhang, Jianhua

    2010-01-01

    CD8α exodomain protein, a crucial immune-system factor in rhesus macaque (M. mulatta), one of the best animal models for vaccine design, was assembled and crystallized. The full structure data will contribute to future studies of immune responses in rhesus macaques. As a T-cell co-receptor, CD8 binds to MHC class I molecules and plays a pivotal role in the activation of cytotoxic T lymphocytes. To date, structures of CD8 have been solved for two different mammals: human and mouse. The infection of rhesus macaques (Macaca mulatta) by simian immunodeficiency virus (SIV) is the best animal model for studying HIV. In this study, the rhesus macaque CD8 (rCD8) αα homodimer was obtained and rCD8α exodomain protein crystals were successfully obtained for further structural analysis. Diffraction data were collected to a resolution of 2.4 Å. The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.52, b = 56.28, c = 82.40 Å. These data will facilitate further studies on the structural differences between these CD8 structures and the cellular immune responses of rhesus macaque

  18. Preparation, crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC1692 from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Chin, Ko-Hsin; Huang, Zhao-Wei; Wei, Kun-Chou; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A conserved hypothetical protein XC1692 from X. campestris pv. campestris has been overexpressed in E. coli. The purified recombinant protein crystallized in a variety of forms and diffracted to a resolution of at least 1.45 Å. Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 Å resolution. They are hexagonal and belong to space group P6 3 , with unit-cell parameters a = b = 56.9, c = 71.0 Å. They contain one molecule per asymmetric unit

  19. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    International Nuclear Information System (INIS)

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-01-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55, b = 90, c = 96 Å

  20. Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

    International Nuclear Information System (INIS)

    Vivan, Ana Luiza; Dias, Márcio Vinícius Bertacini; Schneider, Cristopher Z.; Azevedo, Walter Filgueira Jr de; Basso, Luiz Augusto; Santos, Diógenes Santiago

    2006-01-01

    The M. tuberculosis prephenate dehydratase was cloned, expressed, purified, crystallized by the hanging-drop vapour-diffusion method, and a complete data set collected to 3.2 Å resolution using synchrotron radiation. These results should pave the way for the three-dimensional structure determination of the enzyme and provide a framework on which to base the rational design of chemotherapeutic agents to treat tuberculosis. Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 Å resolution using a synchrotron-radiation source

  1. Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen

    International Nuclear Information System (INIS)

    Kezuka, Yuichiro; Itagaki, Takashi; Satoh, Rie; Teshima, Reiko; Nonaka, Takamasa

    2009-01-01

    A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. An immunologically active mutant of BWp16 was prepared and a three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled mutant protein. A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16ΔN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16ΔN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 Å, α = 111.92, β = 108.91, γ = 98.74°. One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 Å 3 Da −1

  2. Crystallization and preliminary X-ray diffraction analysis of YisP protein from Bacillus subtilis subsp. subtilis strain 168

    International Nuclear Information System (INIS)

    Hu, Yumei; Jia, Shiru; Ren, Feifei; Huang, Chun-Hsiang; Ko, Tzu-Ping; Mitchell, Douglas A.; Guo, Rey-Ting; Zheng, Yingying

    2012-01-01

    A bacteria biofilm formation involved enzyme, BsYisP, from Bacillus subtilis subsp. subtilis strain 168, was crystallized and diffracted to 1.92 Å. YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Å resolution. Structure determination using MAD and MIR methods is in progress

  3. Heterologous expression, purification, crystallization and preliminary X-ray analysis of Trichoderma reesei xylanase II and four variants

    International Nuclear Information System (INIS)

    Wan, Qun; Kovalevsky, Andrey; Zhang, Qiu; Hamilton-Brehm, Scott; Upton, Rosalynd; Weiss, Kevin L.; Mustyakimov, Marat; Graham, David; Coates, Leighton; Langan, Paul

    2013-01-01

    The wild-type protein and four active-site mutants of xylanase II from Trichoderma reesei that catalyzes the hydrolysis of glycosidic bonds in xylan have successfully been crystallized. The crystallization of several structures including ligand-free and protein ligand complexes containing the substrate (xylohexaose) or product (xylotriose) are detailed. Xylanase II from Trichoderma reesei catalyzes the hydrolysis of glycosidic bonds in xylan. Crystallographic studies of this commercially important enzyme have been initiated to investigate its reaction mechanism, substrate binding and dependence on basic pH conditions. The wild-type protein was heterologously expressed in an Escherichia coli host using the defined medium and four active-site amino acids were replaced to abolish its activity (E177Q and E86Q) or to change its pH optimum (N44D and N44H). Cation-exchange and size-exclusion chromatography were used to obtain >90% protein purity. The ligand-free proteins and variant complexes containing substrate (xylohexaose) or product (xylotriose) were crystallized in several different space groups and diffracted to high resolutions (from 1.07 to 1.55 Å)

  4. Crystallization and preliminary X-ray crystallographic analysis of human RGS10 complexed with Gαi3

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyung Ki; Rhee, Kyung Hee [Life Sciences Division, Korea Institute of Science and Technology, Seoul 130-650 (Korea, Republic of); School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Chan-Wha [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Hwang, Kwang Yeon; Kim, Eunice EunKyeong, E-mail: eunice@kist.re.kr [Life Sciences Division, Korea Institute of Science and Technology, Seoul 130-650 (Korea, Republic of)

    2005-09-01

    Human RGS10 and Gαi3 have been overexpressed and purified. Their complex has been crystallized (space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, unit-cell parameters a = 99.88, b = 99.88, c = 144.59 Å, α = β = γ = 90°) and diffraction data have been collected to 2.5 Å resolution using synchrotron radiation. G-protein-coupled receptors, which are major targets for drug discovery, play a major role in diverse physiological processes by relating changes in the extracellular environment to intracellular functions via activation of heterotrimeric G-proteins. However, G-protein activity is also modulated by a family of proteins called regulators of G-protein signalling (RGS), which are classified into six subfamilies. RGS10 belongs to the subgroup D/R12 and is known to act specifically on activated forms of three Gα proteins (Gαi3, Gαz and Gαo but not Gαs). It is abundantly expressed in brain and immune tissues and has been implicated in the pathophysiology of schizophrenia. The RGS domain of RGS10 was cloned, purified, complexed with human Gαi3 and crystallized. The crystals containing both RGS and Gαi3 belong to space group P4{sub 3}2{sub 1}2 (or P4{sub 1}2{sub 1}2), with unit-cell parameters a = 99.88, b = 99.88, c = 144.59 Å, α = β = γ = 90°. A full set of diffraction data were collected to 2.5 Å resolution at 100 K using synchrotron radiation at Pohang beamline 4A.

  5. Crystallization and preliminary X-ray crystallographic analysis of human RGS10 complexed with Gαi3

    International Nuclear Information System (INIS)

    Lee, Hyung Ki; Rhee, Kyung Hee; Kim, Chan-Wha; Hwang, Kwang Yeon; Kim, Eunice EunKyeong

    2005-01-01

    Human RGS10 and Gαi3 have been overexpressed and purified. Their complex has been crystallized (space group P4 3 2 1 2 or P4 1 2 1 2, unit-cell parameters a = 99.88, b = 99.88, c = 144.59 Å, α = β = γ = 90°) and diffraction data have been collected to 2.5 Å resolution using synchrotron radiation. G-protein-coupled receptors, which are major targets for drug discovery, play a major role in diverse physiological processes by relating changes in the extracellular environment to intracellular functions via activation of heterotrimeric G-proteins. However, G-protein activity is also modulated by a family of proteins called regulators of G-protein signalling (RGS), which are classified into six subfamilies. RGS10 belongs to the subgroup D/R12 and is known to act specifically on activated forms of three Gα proteins (Gαi3, Gαz and Gαo but not Gαs). It is abundantly expressed in brain and immune tissues and has been implicated in the pathophysiology of schizophrenia. The RGS domain of RGS10 was cloned, purified, complexed with human Gαi3 and crystallized. The crystals containing both RGS and Gαi3 belong to space group P4 3 2 1 2 (or P4 1 2 1 2), with unit-cell parameters a = 99.88, b = 99.88, c = 144.59 Å, α = β = γ = 90°. A full set of diffraction data were collected to 2.5 Å resolution at 100 K using synchrotron radiation at Pohang beamline 4A

  6. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae

    International Nuclear Information System (INIS)

    Brillet, Karl; Meksem, Ahmed; Thompson, Andrew; Cobessi, David

    2009-01-01

    ShuA from S. dysenteriae was crystallized in several crystallization conditions containing detergents. Adding heavy atoms during crystallization strongly improved the crystal quality and the resolution limits. Diffraction data were collected at an energy remote from the Pb M absorption edges. As part of efforts towards understanding the crystallization of membrane proteins and membrane transport across the outer membrane of Gram-negative bacteria, the TonB-dependent haem outer membrane transporter ShuA of Shigella dysenteriae bound to heavy atoms was crystallized in several crystallization conditions using detergents. The insertion of a His 6 tag into an extracellular loop of ShuA, instead of downstream of the Escherichia coli peptide signal, allowed efficient targeting to the outer membrane and the rapid preparation of crystallizable protein. Crystals diffracting X-rays beyond 3.5 Å resolution were obtained by co-crystallizing ShuA with useful heavy atoms for phasing (Eu, Tb, Pb) by the MAD method at the synchrotron, and the SAD or SIRAS method at the Cu wavelength. The authors collected X-ray diffraction data at 2.3 Å resolution using one crystal of ShuA-Pb, and at 3.2 Å resolution at an energy remote from the Pb M absorption edges for phasing on PROXIMA-1 at SOLEIL

  7. Crystallization and preliminary X-ray crystallographic analysis of CheW from Thermotoga maritima: a coupling protein of CheA and the chemotaxis receptor

    International Nuclear Information System (INIS)

    Park, SangYoun; Crane, Brian R.

    2011-01-01

    CheW from T. maritima has been crystallized (space group P6 3 , unit-cell parameters a = b = 61.265, c = 361.045 Å). Diffraction data have been collected to 3.1 Å resolution using synchrotron X-ray radiation. The CheW protein plays a key role in bacterial chemotaxis signal transduction by coupling CheA to chemotaxis receptors. CheW from Thermotoga maritima has been overexpressed in Escherichia coli and crystallized at 298 K using ammonium sulfate as a salt precipitant. X-ray diffraction data have been collected to 3.10 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group P6 3 , with unit-cell parameters a = b = 61.265, c = 361.045 Å. The asymmetric unit may contain four to six CheW molecules

  8. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of variants of monoamine oxidase from Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Atkin, Kate E. [Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Reiss, Renate; Turner, Nicholas J. [School of Chemistry, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN (United Kingdom); Brzozowski, Andrzej M.; Grogan, Gideon, E-mail: grogan@ysbl.york.ac.uk [Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom)

    2008-03-01

    Crystals of A. niger monoamine oxidase variants display P2{sub 1} or P4{sub 1}2{sub 1}2/P4{sub 3}2{sub 1}2 symmetry, with eight or two molecules in the asymmetric unit, respectively. Monoamine oxidase from Aspergillus niger (MAO-N) is an FAD-dependent enzyme that catalyses the conversion of terminal amines to their corresponding aldehydes. Variants of MAO-N produced by directed evolution have been shown to possess altered substrate specificity. Crystals of two of these variants (MAO-N-3 and MAO-N-5) have been obtained; the former displays P2{sub 1} symmetry with eight molecules per asymmetric unit and the latter has P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 symmetry and two molecules per asymmetric unit. Solution of these structures will help shed light on the molecular determinants of improved activity and high enantioselectivity towards a broad range of substrates.

  9. Purification, crystallization and preliminary X-ray analysis of TP0435 (Tp17) from the syphilis spirochete Treponema pallidum

    International Nuclear Information System (INIS)

    Brautigam, Chad A.; Deka, Ranjit K.; Norgard, Michael V.

    2013-01-01

    The soluble portion of TP0435, a putative periplasmic lipoprotein from the syphilis spirochete T. pallidum, has been purified and crystallized in a rhombohedral space group. A complete native data set has been collected to 2.4 Å resolution. Syphilis, caused by the bacterial spirochete Treponema pallidum, remains a prominent sexually transmitted infection worldwide. Despite sequencing of the genome of this obligate human pathogen 15 years ago, the functions of a large number of the gene products of T. pallidum are still unknown, particularly with respect to those of the organism’s periplasmic lipoproteins. To better understand their functions, a structural biology approach has been pursued. To this end, the soluble portion of the T. pallidum TP0435 lipoprotein (also known as Tp17) was cloned, hyper-expressed in Escherichia coli and purified to apparent homogeneity. The protein crystals obtained from this preparation diffracted to 2.4 Å resolution and had the symmetry of space group R3. In the hexagonal setting, the unit-cell parameters were a = b = 85.7, c = 85.4 Å

  10. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Gregg, Katie J.; Boraston, Alisdair B.

    2009-01-01

    The catalytic module of a family 101 glycoside hydrolase from S. pneumoniae was cloned, recombinantly produced and crystallized. Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-d-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2 1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 Å, β = 112.46°. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl) -α-d-galactopyranoside, which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase

  11. Crystallization and preliminary X-ray analysis of 5-keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-d-gluconate and NADPH

    International Nuclear Information System (INIS)

    Kubota, Keiko; Miyazono, Ken-ichi; Nagata, Koji; Toyama, Hirohide; Matsushita, Kazunobu; Tanokura, Masaru

    2010-01-01

    NADPH-dependent 5-keto-d-gluconate reductase from G. suboxydans IFO12528 (5KGR) was expressed, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method. Crystals of the 5KGR–NADPH complex and of the 5KGR–NADPH–5-keto-d-gluconate complex diffracted X-rays to 1.75 and 2.26 Å resolution, respectively. NADPH-dependent 5-keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-d-gluconate and d-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR–NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Å resolution. The crystal of the complex belonged to space group P4 2 2 1 2, with unit-cell parameters a = b = 128.6, c = 62.9 Å. A crystal of the 5KGR–NADPH–5-keto-d-gluconate complex was prepared by soaking the 5KGR–NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-d-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P4 2 2 1 2, with unit-cell parameters a = b = 128.7, c = 62.5 Å. Both crystals contained two molecules in the asymmetric unit

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    International Nuclear Information System (INIS)

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-01-01

    The carbohydrate-binding component (VP8* 64–223 ) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8* 64–223 structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3 2 21 and monoclinic P2 1 ) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8* 64–223 structure by molecular replacement

  13. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    Energy Technology Data Exchange (ETDEWEB)

    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  14. Crystallization of uridine phosphorylase from Shewanella oneidensis MR-1 in the laboratory and under microgravity and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Safonova, Tatyana N.; Mordkovich, Nadezhda N.; Polyakov, Konstantin M.; Manuvera, Valentin A.; Veiko, Vladimir P.; Popov, Vladimir O.

    2012-01-01

    High-quality crystals of uridine phosphorylase from Shewanella oneidensis were grown under microgravity conditions. X-ray diffraction data were collected to a resolution of 0.95 Å. Uridine phosphorylase (UDP, EC 2.4.2.3), a key enzyme in the pyrimidine salvage pathway, catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The gene expression of UDP from Shewanella oneidensis MR-1 was performed in the recipient strain Escherichia coli. The UDP protein was crystallized on earth (in the free form and in complex with uridine as the substrate) by the hanging-drop vapour-diffusion method at 296 K and under microgravity conditions (in the free form) aboard the Russian Segment of the International Space Station by the capillary counter-diffusion method. The data sets were collected to a resolution of 1.9 Å from crystals of the free form grown on earth, 1.6 Å from crystals of the complex with uridine and 0.95 Å from crystals of the free form grown under microgravity. All crystals belong to the space group P2 1 and have similar unit-cell parameters. The crystal of uridine phosphorylase grown under microgravity diffracted to ultra-high resolution and gave high-quality X-ray diffraction data

  15. Purification, crystallization and preliminary X-ray analysis of l-sorbose reductase from Gluconobacter frateurii complexed with l-sorbose or NADPH

    International Nuclear Information System (INIS)

    Kubota, Keiko; Nagata, Koji; Miyazono, Ken-ichi; Toyama, Hirohide; Matsushita, Kazunobu; Tanokura, Masaru

    2009-01-01

    NADPH-dependent l-sorbose reductase from G. frateurii (SR) was expressed, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method. Crystals of the SR–l-sorbose complex and SR–NADPH complex diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. NADPH-dependent l-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR–l-sorbose complex and the SR–NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. The crystal of the SR–l-sorbose complex belonged to space group C222 1 , with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 Å. The crystal of the SR–NADPH complex belonged to space group P2 1 , with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 Å, β = 89.99°. The crystals contained two and eight molecules, respectively, in the asymmetric unit

  16. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708

    International Nuclear Information System (INIS)

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    Conjugated polyketone reductase C2 from C. parapsilosis IFO 0708 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystal belonged to space group P2 1 2 1 2 1 and diffracted X-rays to 1.7 Å resolution. Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 Å resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 Å. The Matthews coefficient (V M = 1.76 Å 3 Da −1 ) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit

  17. Crystallization and preliminary X-ray diffraction analysis of CfaE, the adhesive subunit of the CFA/I fimbriae from human enterotoxigenic Escherichia coli

    International Nuclear Information System (INIS)

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; Esser, Lothar; Savarino, Stephen J.; Xia, Di

    2006-01-01

    The adhesin CfaE of the CFA/I fimbriae from human enterotoxigenic E. coli has been crystallized. CfaE crystals diffracted X-rays to better than 2.4 Å and phasing was solved by the SIRAS method. Enterotoxigenic Escherichia coli (ETEC) represents a formidable food and waterborne diarrheal disease threat of global importance. The first step in ETEC pathogenesis is bacterial attachment to small-intestine epithelial cells via adhesive fimbriae, many of which are genetically related to the prototype colonization factor antigen I (CFA/I). The minor fimbrial subunit CfaE is required for initiation of CFA/I fimbrial assembly and mediates bacterial attachment to host cell-surface receptors. A donor-strand complemented variant of CfaE (dscCfaE) was expressed with a hexahistidine tag, purified to homogeneity and crystallized using the hanging-drop vapor-diffusion method. X-ray diffraction data sets were collected to 2.4 Å resolution for both native and derivatized crystals and showed the symmetry of space group P6 2 22, with unit-cell parameters a = b = 142.9, c = 231.9 Å. Initial phases were derived from the SIRAS approach and electron density showed two molecules in the crystallographic asymmetric unit. Sequence assignments were aided by anomalous signals from the selenium of an SeMet-derivatized crystal and from S atoms of a native crystal

  18. Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acid

    International Nuclear Information System (INIS)

    Santos, Juliana I. dos; Santos-Filho, Norival A.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2010-01-01

    PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A 2 from B. pirajai venom, was cocrystallized with the inhibitor rosmarinic acid from C. verbenacea. The crystals diffracted X-rays to 1.8 Å resolution and the structure was solved, indicating a remarkable electronic density for the ligand at the entrance to the hydrophobic channel. PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A 2 from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 Å resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2 1 2 1 2 1 , indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A 2 structures belong to space group P3 1 21, while the crystals of complexed structures belong to space groups P2 1 or P2 1 2 1 2 1

  19. Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A{sub 2} homologue from Bothrops pirajai venom complexed with rosmarinic acid

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Juliana I. dos [Departamento de Física e Biofísica, Instituto de Biociências, UNESP - Universidade Estadual Paulista, Botucatu-SP (Brazil); Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq (Brazil); Santos-Filho, Norival A.; Soares, Andreimar M. [Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq (Brazil); Departamento de Análizes Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP, Ribeirão Preto-SP (Brazil); Fontes, Marcos R. M., [Departamento de Física e Biofísica, Instituto de Biociências, UNESP - Universidade Estadual Paulista, Botucatu-SP (Brazil); Instituto Nacional de Ciência e Tecnologia em Toxinas, CNPq (Brazil)

    2010-06-01

    PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A{sub 2} from B. pirajai venom, was cocrystallized with the inhibitor rosmarinic acid from C. verbenacea. The crystals diffracted X-rays to 1.8 Å resolution and the structure was solved, indicating a remarkable electronic density for the ligand at the entrance to the hydrophobic channel. PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A{sub 2} from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 Å resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A{sub 2} structures belong to space group P3{sub 1}21, while the crystals of complexed structures belong to space groups P2{sub 1} or P2{sub 1}2{sub 1}2{sub 1}.

  20. Cloning, expression, purification, crystallization and preliminary X-ray analysis of NodS N-methyltransferase from Bradyrhizobium japonicum WM9

    International Nuclear Information System (INIS)

    Cakici, Ozgur; Sikorski, Michal; Stepkowski, Tomasz; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-01-01

    The NodS N-methyltransferase, an enzyme participating in the biosynthesis of the bacterial nodulation (Nod) factor necessary to establish symbiotic nitrogen fixation with a legume plant host, has been crystallized in the apo form as well as in complex with SAH. SAH is a byproduct of SAM degradation during the SAM-dependent methylation reaction. The Nod factor (NF) is a rhizobial signal molecule that is involved in recognition of a legume host and the formation of root and stem nodules. Some unique enzymes are involved in the biosynthesis of NF, which is a variously but specifically substituted lipochitooligosaccharide. One of these enzymes is NodS, an N-methyltransferase that methylates end-deacetylated chitooligosaccharide substrates. In the methylation reaction, NodS uses S-adenosyl-l-methionine (SAM) as a methyl donor. To date, no structural information is available about NodS from any rhizobium. X-ray crystallographic studies of the NodS protein from Bradyrhizobium japonicum WM9, which infects the legumes lupin and serradella, have been undertaken. The nodS gene was cloned and the recombinant protein was expressed in Escherichia coli cells using natural amino acids and as an SeMet derivative. NodS without ligands was crystallized in the presence of PEG 3350 and MgCl 2 . The protein was also crystallized in complex with S-adenosyl-l-homocysteine (SAH) in the presence of PEG 8000 and MgCl 2 . SAH is produced from SAM as a byproduct of the methylation reaction. The crystals of apo NodS are tetragonal and diffracted X-rays to 2.42 Å resolution. The NodS–SAH complex crystallizes in an orthorhombic space group and the crystals diffracted X-rays to 1.85 Å resolution

  1. Purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal fragment of the MvfR protein from Pseudomonas aeruginosa

    International Nuclear Information System (INIS)

    Kefala, Katerina; Kotsifaki, Dina; Providaki, Mary; Kapetaniou, Evangelia G.; Rahme, Lawrence; Kokkinidis, Michael

    2012-01-01

    MvfRC87, a 242-residue C-terminal segment of the LysR-type transcriptional regulator MvfR, was produced in Escherichia coli, purified and crystallized. The LysR-type transcriptional regulator MvfR plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing-regulated virulence factors. The protein also controls pathogenic type VI secretion loci. MvfRC87, a 242-residue C-terminal segment of MvfR, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected using synchrotron radiation and crystallographic parameters were determined

  2. Cloning, purification, crystallization and preliminary X-ray diffraction analysis of nitrile hydratase from the themophilic Bacillus smithii SC-J05-1

    International Nuclear Information System (INIS)

    Hourai, Shinji; Ishii, Takeshi; Miki, Misao; Takashima, Yoshiki; Mitsuda, Satoshi; Yanagi, Kazunori

    2005-01-01

    The nitrile hydratase from the themophilic B. smithii SC-J05-1 (Bs NHase) has been purified, cloned and crystallized. Nitrile hydratase (NHase) converts nitriles to the corresponding amides and is recognized as having important industrial applications. Purification, cloning, crystallization and initial crystallographic studies of the NHase from Bacillus smithii SC-J05-1 (Bs NHase) were conducted to analyze the activity, specificity and thermal stability of this hydrolytic enzyme. Bs NHase was purified to homogeneity from microbial cells of B. smithii SC-J05-1 and the nucleotide sequences of both the α- and β-subunits were determined. Purified Bs NHase was used for crystallization and several crystal forms were obtained by the vapour-diffusion method. Microseeding and the addition of magnesium ions were essential components to obtain crystals suitable for X-ray diffraction analysis

  3. Crystallization and preliminary X-ray diffraction study of recombinant adenine phosphoribosyltransferase from the thermophilic bacterium Thermus thermophilus strain HB27

    Science.gov (United States)

    Sinitsyna, E. V.; Timofeev, V. I.; Tuzova, E. S.; Kostromina, M. A.; Murav'eva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2017-07-01

    Adenine phosphoribosyltransferase (APRT) belongs to the type I phosphoribosyltransferase family and catalyzes the formation of adenosine monophosphate via transfer of the 5-phosphoribosyl group from phosphoribosyl pyrophosphate to the nitrogen atom N9 of the adenine base. Proteins of this family are involved in a salvage pathway of nucleotide synthesis, thus providing purine base utilization and maintaining the optimal level of purine bases in the body. Adenine phosphoribosyltransferase from the extremely thermophilic Thermus thermophilus strain HB27 was produced using a highly efficient E. coli producer strain and was then purified by affinity and gel-filtration chromatography. This enzyme was successfully employed as a catalyst for the cascade biosynthesis of biologically important nucleotides. The screening of crystallization conditions for recombinant APRT from T. thermophilus HB27 was performed in order to determine the enzyme structure by X-ray diffraction. The crystallization conditions, which were found by the vapor-diffusion technique, were then optimized to apply the counter-diffusion technique. The crystals of the enzyme were grown by the capillary counter-diffusion method. The crystals belong to sp. gr. P1211 and have the following unitcell parameters: a = 69.86 Å, b = 82.16 Å, c = 91.39 Å, α = γ = 90°, β = 102.58°. The X-ray diffraction data set suitable for the determination of the APRT structure at 2.6 Å resolution was collected from the crystals at the SPring-8 synchrotron facility (Japan).

  4. Overexpression, crystallization and preliminary X-ray crystallographic analysis of Pseudomonas aeruginosa MnmE, a GTPase involved in tRNA modification

    International Nuclear Information System (INIS)

    Lee, Hyung Ho; Suh, Se Won

    2010-01-01

    MnmE from P. aeruginosa was crystallized and X-ray diffraction data were collected to 2.69 Å resolution. MnmE, an evolutionarily conserved GTPase, is involved in modification of the uridine base (U34) at the wobble position of certain tRNAs. Previous crystal structures of MnmE suggest that it is a dimer with considerable conformational flexibility. To facilitate structural comparisons among MnmE proteins, structural analysis of MnmE from Pseudomonas aeruginosa encoded by the PA5567 gene was initiated. It was overexpressed in Escherichia coli and crystallized at 297 K using a reservoir solution consisting of 100 mM sodium ADA pH 6.5, 12%(w/v) polyethylene glycol 4000, 100 mM lithium sulfate, 2%(v/v) 2-propanol and 2.5 mM dithiothreitol. X-ray diffraction data were collected to 2.69 Å resolution. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 96.74, b = 204.66, c = 120.90 Å. Two monomers were present in the asymmetric unit, resulting in a crystal volume per protein mass (V M ) of 2.99 Å 3 Da −1 and a solvent content of 58.8%

  5. SECCOX, a novel x-ray characterization bench for Bragg crystals and x-rays optics

    International Nuclear Information System (INIS)

    Caillaud, T.; Manson, M.; Desenne, D.; Goze, B.; Rivet, A.; Derouineau, Ph.

    2007-01-01

    Laser programs require the use of a large number of calibrated x-ray crystals implemented inside spectrometers and microscopes used in diagnostics. In this context, a new apparatus was designed in collaboration with CELIA laboratory, Saint-Gobain Crystals and Detectors and CEA to characterize x-ray Bragg crystals. Station d'Etude et de Caracterisation des Cristaux pour les Optiques X (SECCOX) is based on a micrometric x-ray source and an automated spectrometer equipped with a CCD camera. Properties such as homogeneity, resolution, radius of curvature and reflectivity are measured to guarantee diagnostic performance in laser-plasma physics experiments. We will present the experimental device, techniques and results of the calibration obtained. (authors)

  6. Expression, purification, crystallization and preliminary X-ray analysis of conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708.

    Science.gov (United States)

    Yamamura, Akihiro; Maruoka, Shintaro; Ohtsuka, Jun; Miyakawa, Takuya; Nagata, Koji; Kataoka, Michihiko; Kitamura, Nahoko; Shimizu, Sakayu; Tanokura, Masaru

    2009-11-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708 is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily and catalyzes the stereospecific reduction of ketopantoyl lactone to d-pantoyl lactone. A diffraction-quality crystal of recombinant CPR-C2 was obtained by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.7 angstrom resolution on beamline NW12A of the Photon Factory-Advanced Ring (Tsukuba, Japan). The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.02, b = 68.30, c = 68.93 angstrom. The Matthews coefficient (V(M) = 1.76 angstrom(3) Da(-1)) indicated that the crystal contained one CPR-C2 molecule per asymmetric unit.

  7. Crystallization and preliminary X-ray analysis of reducing-end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125

    International Nuclear Information System (INIS)

    Honda, Yuji; Fushinobu, Shinya; Hidaka, Masafumi; Wakagi, Takayoshi; Shoun, Hirofumi; Kitaoka, Motomitsu

    2005-01-01

    Reducing-end-xylose releasing exo-oligoxylanase (Rex) from B. halodurans C-125 was crystallized. A diffraction data set was collected to 1.35 Å resolution. The reducing-end xylose-releasing exo-oligoxylanase (Rex) from Bacillus halodurans C-125, a novel family GH8 glycoside hydrolase, was crystallized by the hanging-drop vapour-diffusion method using 13.6 mg ml −1 purified Rex, 5.6%(v/v) polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30%(v/v) glycerol. Suitable crystals grew after incubation for 5 d at 293 K. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 Å. X-ray diffraction data were collected at a resolution of 1.35 Å

  8. Crystallization and preliminary X-ray analysis of the complex of NADH and 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Sachiyo [Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522 (Japan); Nakamura, Shota; Ohkubo, Tadayasu [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ueda, Shigeru [Diagnostics Department, Asahi Kasei Pharma Corporation, 632-1 Mifuku, Izunokuni, Shizuoka 410-2321 (Japan); Uchiyama, Susumu [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kobayashi, Yuji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Oda, Masayuki, E-mail: oda@kpu.ac.jp [Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522 (Japan)

    2006-06-01

    The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 has been crystallized and X-ray diffraction data have been collected to 1.8 Å resolution. The NAD(P){sup +}-dependent enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3α-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 Å. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 Å, and contained two molecules, reflecting dimer formation of 3α-HSD, in the asymmetric unit.

  9. Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii

    International Nuclear Information System (INIS)

    Tonkin, Michelle L.; Brown, Shannon; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

    2012-01-01

    To characterize the ISP family of proteins present in apicomplexan parasites, ISP1 from T. gondii was expressed, purified and crystallized. Two crystal forms (cubic and orthorhombic) were analyzed by X-ray diffraction and data were processed to 2.05 and 2.1 Å resolution, respectively. The protozoan parasites of the Apicomplexa phylum are devastating global pathogens. Their success is largely due to phylum-specific proteins found in specialized organelles and cellular structures. The inner membrane complex (IMC) is a unique apicomplexan structure that is essential for motility, invasion and replication. The IMC subcompartment proteins (ISP) have recently been identified in Toxoplasma gondii and shown to be critical for replication, although their specific mechanisms are unknown. Structural characterization of TgISP1 was pursued in order to identify the fold adopted by the ISPs and to generate detailed insight into how this family of proteins functions during replication. An N-terminally truncated form of TgISP1 was purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis. Two crystal forms of TgISP1 belonging to space groups P4 1 32 or P4 3 32 and P2 1 2 1 2 1 diffracted to 2.05 and 2.1 Å resolution, respectively

  10. Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF

    International Nuclear Information System (INIS)

    Nomoto, Ryohei; Tezuka, Takeaki; Miyazono, Ken-ichi; Tanokura, Masaru; Horinouchi, Sueharu; Ohnishi, Yasuo

    2012-01-01

    A Streptomyces homologue of the mycobacterial integration host factor mIHF was heterologously produced, purified and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.22 Å resolution and belonged to space group C2. The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution

  11. Purification, crystallization and preliminary X-ray analysis of SGR6054, a Streptomyces homologue of the mycobacterial integration host factor mIHF

    Energy Technology Data Exchange (ETDEWEB)

    Nomoto, Ryohei; Tezuka, Takeaki; Miyazono, Ken-ichi; Tanokura, Masaru; Horinouchi, Sueharu; Ohnishi, Yasuo [Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2012-08-31

    A Streptomyces homologue of the mycobacterial integration host factor mIHF was heterologously produced, purified and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The best crystal diffracted X-rays to 2.22 Å resolution and belonged to space group C2. The mycobacterial integration host factor (mIHF) is a small nonspecific DNA-binding protein that is essential for the growth of Mycobacterium smegmatis. mIHF homologues are widely distributed among Actinobacteria, and a Streptomyces homologue of mIHF is involved in control of sporulation and antibiotic production in S. coelicolor A3(2). Despite their important biological functions, a structure of mIHF or its homologues has not been elucidated to date. Here, the S. griseus mIHF homologue (SGR6054) was expressed and purified from Escherichia coli and crystallized in the presence of a 16-mer duplex DNA by the sitting-drop vapour-diffusion method. The plate-shaped crystal belonged to space group C2, with unit-cell parameters a = 88.53, b = 69.35, c = 77.71 Å, β = 96.63°, and diffracted X-rays to 2.22 Å resolution.

  12. Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

    Science.gov (United States)

    Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R; Jauch, Ralf

    2014-10-01

    Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

  13. Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element

    Science.gov (United States)

    Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R.; Jauch, Ralf

    2014-01-01

    Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6–DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit. PMID:25286939

  14. Crystallization and preliminary X-ray diffraction analysis of the Fab fragment of WO2, an antibody specific for the Aβ peptides associated with Alzheimer’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Wun, Kwok S. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Miles, Luke A. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Crespi, Gabriela A. N. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Wycherley, Kaye [WEHI Biotechnology Centre, La Trobe R& D Park, Bundoora, Victoria 3086 (Australia); Ascher, David B. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Barnham, Kevin J.; Cappai, Roberto [Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Beyreuther, Konrad [ZMBH, University of Heidelberg, Heidelberg (Germany); Masters, Colin L. [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); The Mental Health Research Institute of Victoria, Parkville, Victoria 3052 (Australia); Parker, Michael W. [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010 (Australia); McKinstry, William J., E-mail: wjmckinstry@hotmail.com [Biota Structural Biology Laboratory and Centre for Structural Neurobiology, St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065 (Australia); Department of Medicine (St Vincent’s Hospital), The University of Melbourne, 41 Victoria Parade, Fitzroy 3065 (Australia)

    2008-05-01

    Crystallization and X-ray diffraction data collection of the Fab fragment of the monoclonal antibody WO2 in the absence or presence of amyloid β peptides associated with Alzheimer’s disease are reported. The murine monoclonal antibody WO2 specifically binds the N-terminal region of the amyloid β peptide (Aβ) associated with Alzheimer’s disease. This region of Aβ has been shown to be the immunodominant B-cell epitope of the peptide and hence is considered to be a basis for the development of immunotherapeutic strategies against this prevalent cause of dementia. Structural studies have been undertaken in order to characterize the molecular basis for antibody recognition of this important epitope. Here, details of the crystallization and X-ray analysis of the Fab fragment of the unliganded WO2 antibody in two crystal forms and of the complexes that it forms with the truncated Aβ peptides Aβ{sub 1–16} and Aβ{sub 1–28} are presented. These crystals were all obtained using the hanging-drop vapour-diffusion method at 295 K. Crystals of WO2 Fab were grown in polyethylene glycol solutions containing ZnSO{sub 4}; they belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1} and diffracted to 1.6 Å resolution. The complexes of WO2 Fab with either Aβ{sub 1–@}@{sub 16} or Aβ{sub 1–28} were cocrystallized from polyethylene glycol solutions. These two complex crystals grew in the same space group, P2{sub 1}2{sub 1}2{sub 1}, and diffracted to 1.6 Å resolution. A second crystal form of WO2 Fab was grown in the presence of the sparingly soluble Aβ{sub 1–42} in PEG 550 MME. This second form belonged to space group P2{sub 1} and diffracted to 1.9 Å resolution.

  15. Crystallization and preliminary x-ray structure analysis of the egg-white lysozyme from a Taiwanese Soft-Shelled Turtle (Tri onyx Sinensis Wiegmann)

    International Nuclear Information System (INIS)

    Siritapetawee, Jaruwan; Thammasirirak, Sompong; Yuvaniyama, Jirudon

    2005-10-01

    Lysozyme has been purified from the egg-white of a Taiwanese soft-shelled turtle. This soft-shelled turtle ' s egg-white lysozyme migrated on 12.5% SDS-PAGE at about 14.8 kDa. The lysozyme has been crystallized using the sitting drop vapor diffusion technique and 30% (w/v) polyethylene glycol 8000 in 0.1 M sodium cacodylate, p H 6.5 containing 0.2 M ammonium sulfate as a precipitant. One of the crystals diffracted X rays beyond 2 angstrom unit resolution and belonged to the orthorhombic, space group P212121, with unit cell dimensions of a = 37.8 angstrom unit, b = 55.6 angstrom unit, and c 72.2 angstrom unit and one molecule of the enzyme per asymmetric unit. The data were collected to 1.9 angstrom resolution with an R merge of 4.6%, suitable for high resolution structure analysis. The single-crystal X-ray structure of lysozyme has been initially phased with the Molecular Replacement technique using pheasant egg-white lysozyme (PDB ID 1GHL) as a search template. Model rebuilding and refinement are in progress

  16. Crystallization and preliminary X-ray crystallographic analysis of Thermotoga maritima CheA P3-P4-P5 domains in complex with CheW

    International Nuclear Information System (INIS)

    Park, SangYoun; Kim, Keon Young; Kim, Sunmin; Crane, Brian R.

    2012-01-01

    T. maritima CheA P3-P4-P5 domains were crystallized in complex with CheW. Low-resolution diffraction data were collected to ∼8 Å using synchrotron X-ray radiation. The CheA–CheW complex plays a key role in bacterial chemotaxis signal transduction by initiating phosphotransfer to response regulators via coupling to the chemoreceptors. CheA (P3-P4-P5 domains) and CheW from Thermotoga maritima were overexpressed in Escherichia coli and crystallized as a complex at 298 K using ammonium dihydrogen phosphate as a precipitant. X-ray diffraction data were collected to ∼8 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 184.2, b = 286.4, c = 327.7 Å. The asymmetric unit may contain six to ten CheA–CheW molecules

  17. Purification, crystallization and preliminary X-ray analysis of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

    Science.gov (United States)

    Deva, Taru; Pryor, KellyAnn D; Leiting, Barbara; Baker, Edward N; Smith, Clyde A

    2003-08-01

    UDP-N-acetylmuramoyl:L-alanine ligase (MurC) is involved in the pathway leading from UDP-N-glucosamine to the UDP-N-acetylmuramoyl:pentapeptide unit, which is the building block for the peptidoglycan layer found in all bacterial cell walls. The pathways leading to the biosynthesis of the peptidoglycan layer are important targets for the development of novel antibiotics, since animal cells do not contain these pathways. MurC is the first of four similar ATP-dependent amide-bond ligases which share primary and tertiary structural similarities. The crystal structures of three of these have been determined by X-ray crystallography, giving insights into the binding of the carbohydrate substrate and the ATP. Diffraction-quality crystals of the enzyme MurC have been obtained in both native and selenomethionine forms and X-ray diffraction data have been collected at the Se edge at a synchrotron source. The crystals are orthorhombic, with unit-cell parameters a = 73.9, b = 93.6, c = 176.8 A, and diffraction has been observed to 2.6 A resolution.

  18. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum {beta}-lactamase conferring severe antibiotic resistance

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J. H., E-mail: msgjhlee@mju.ac.kr; Sohn, S. G., E-mail: sgsohn@mju.ac.kr; Jung, H. I., E-mail: jhinumber1@hanmail.net; An, Y. J., E-mail: anyj0120@hanmail.net; Lee, S. H., E-mail: sangheelee@mju.ac.kr [Myongji University, Drug Resistance Proteomics Laboratory, Department of Biological Sciences (Korea, Republic of)

    2013-07-15

    OXA-17, an extended-spectrum {beta}-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates {beta}-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino {beta}-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 A resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 A. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

  19. Overexpression, crystallization and preliminary X-ray crystallographic analysis of the RNA polymerase domain of primase from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Im, Dong-Won; Kim, Tae-O; Jung, Ha Yun; Oh, Ji Eun; Lee, Se Jin; Heo, Yong-Seok

    2011-01-01

    The RNA polymerase domain of primase from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 1.60 Å. Primase is the enzyme that synthesizes RNA primers on single-stranded DNA during normal DNA replication. In this study, the catalytic core domain of primase from Streptococcus mutans UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 1.60 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P4 1 or P4 3 , with unit-cell parameters a = b = 52.63, c = 110.31 Å. The asymmetric unit is likely to contain one molecule, with a corresponding V M of 1.77 Å 3 Da −1 and a solvent content of 30.7%

  20. Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Paterson, Neil G.; Riboldi-Tunnicliffe, Alan; Mitchell, Timothy J.; Isaacs, Neil W.

    2006-01-01

    The mature form of RafE has been expressed, purified and crystallized. X-ray diffraction data have been collected to 3.65 and 2.90 Å resolution from native and selenomethionine-derivative crystals, respectively. Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23. A truncated form (residues 24–419) of RafE from S. pneumoniae was cloned and overexpressed in Escherichia coli. Native and selenomethionine-labelled protein have been crystallized in the hexagonal space group P6 1 22. Diffraction data have been successfully phased to 2.90 Å using Se SAD data and model building is in progress

  1. Purification, crystallization and preliminary X-ray diffraction analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus mutans strain UA159

    International Nuclear Information System (INIS)

    Kim, Tae-O; Im, Dong-Won; Jung, Ha Yun; Kwon, Seong Jung; Heo, Yong-Seok

    2012-01-01

    Enoyl-acyl carrier protein reductase (FabK) from S. mutans strain UA159 was cloned, overexpressed, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.40 Å. A triclosan-resistant flavoprotein termed FabK is the sole enoyl-acyl carrier protein reductase in Streptococcus pneumoniae and Streptococcus mutans. In this study, FabK from S. mutans strain UA159 was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.40 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P6 2 , with unit-cell parameters a = b = 105.79, c = 44.15 Å. The asymmetric unit contained one molecule, with a corresponding V M of 2.05 Å 3 Da −1 and a solvent content of 39.9%

  2. Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum β-lactamase conferring severe antibiotic resistance

    International Nuclear Information System (INIS)

    Lee, J. H.; Sohn, S. G.; Jung, H. I.; An, Y. J.; Lee, S. H.

    2013-01-01

    OXA-17, an extended-spectrum β-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates β-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino β-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 Å resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%

  3. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando, E-mail: xalbert@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física ‘Rocasolano’, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid (Spain)

    2007-07-01

    Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å.

  4. Crystallization and preliminary X-ray diffraction studies of NP24-I, an isoform of a thaumatin-like protein from ripe tomato fruits

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Raka; Chakrabarti, Chandana, E-mail: chandana.chakrabarti@saha.ac.in [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata 700064 (India)

    2005-08-01

    A thaumatin-like antifungal protein, NP24-I, has been isolated from ripe tomato fruits. It was crystallized by the vapour-diffusion method and data were collected to 2.45 Å. The structure was solved by molecular replacement. NP24 is a 24 kDa (207-amino-acid) antifungal thaumatin-like protein (TLP) found in tomato fruits. An isoform of the protein, NP24-I, is reported to play a possible role in ripening of the fruit in addition to its antifungal properties. The protein has been isolated and purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 61.01, c = 62.90 Å and one molecule per asymmetric unit. X-ray diffraction data were processed to a resolution of 2.45 Å and the structure was solved by molecular replacement.

  5. Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei

    International Nuclear Information System (INIS)

    Abd Aziz, Abd Ghani; Sedelnikova, Svetlana E.; Ruzheinikov, Sergey N.; Thorpe, Simon; Mohamed, Rahmah; Nathan, Sheila; Rafferty, John B.; Baker, Patrick J.; Rice, David W.

    2012-01-01

    The receiver domain of a putative response regulator from B. pseudomallei, BPSL0128, has been crystallized in a form suitable for X-ray analysis. bpsl0128, a gene encoding a putative response regulator from Burkholderia pseudomallei strain D286, has been cloned into a pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. The full-length protein is degraded during purification to leave a fragment corresponding to the putative receiver domain, and crystals of this protein that diffracted to beyond 1.75 Å resolution have been grown by the hanging-drop vapour-diffusion technique using PEG 6000 as the precipitant. The crystals belonged to one of the enantiomorphic pair of space groups P3 1 21 and P3 2 21, with unit-cell parameters a = b = 65.69, c = 105.01 Å and either one or two molecules in the asymmetric unit

  6. Purification, crystallization and preliminary X-ray diffraction analysis of RafE, a sugar-binding lipoprotein from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunnicliffe, Alan [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Mitchell, Timothy J. [Division of Infection and Immunity (IBLS), Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Isaacs, Neil W. [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2006-07-01

    The mature form of RafE has been expressed, purified and crystallized. X-ray diffraction data have been collected to 3.65 and 2.90 Å resolution from native and selenomethionine-derivative crystals, respectively. Streptococcus pneumoniae contains a large number of sugar-transport systems and the system responsible for raffinose uptake has recently been identified. The substrate-binding protein component of this system shares strong sequence homology with the multiple sugar metabolism substrate-binding protein MsmE from S. mutans and contains a lipoprotein-attachment site at cysteine residue 23. A truncated form (residues 24–419) of RafE from S. pneumoniae was cloned and overexpressed in Escherichia coli. Native and selenomethionine-labelled protein have been crystallized in the hexagonal space group P6{sub 1}22. Diffraction data have been successfully phased to 2.90 Å using Se SAD data and model building is in progress.

  7. Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae

    International Nuclear Information System (INIS)

    Li, Yong-Fu; Poole, Steven; Rasulova, Fatima; McVeigh, Annette L.; Savarino, Stephen J.; Xia, Di

    2009-01-01

    Three fusion proteins were generated in order to resolve the atomic structure of the CFA/I fimbriae of enterotoxigenic E. coli. CfaEB is a fusion of the minor and major CFA/I subunits, while CfaBB and CfaBBB are tandem fusions of two and three repeats, respectively, of the major subunit. Each protein was crystallized and the crystal structures of each of these fusions were determined successively by the molecular-replacement method using the CfaE crystal structure as an initial phasing model. Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1 Å resolution and displayed the symmetry of space group P2 1 . CfaBB exhibited a crystal diffraction limit of 2.3 Å resolution and had the symmetry of space group P2 1 2 1 2. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3 Å resolution. These structures were determined using the molecular-replacement method

  8. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    International Nuclear Information System (INIS)

    Byrnes, Laura J.; Badarau, Adriana; Vakulenko, Sergei B.; Smith, Clyde A.

    2008-01-01

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl 2 , 0.1 M Tris–HCl pH 8.5 and 1 mM Mg 2 GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA

  9. Purification, crystallization and preliminary X-ray crystallographic analysis of the ETS domain of human Ergp55 in complex with the cfos promoter DNA sequence

    International Nuclear Information System (INIS)

    Gangwar, Shanti P.; Meena, Sita R.; Saxena, Ajay K.

    2012-01-01

    The ETS domain of human Ergp55 was purified and crystallized in native, complexes with E74, and cfos promoter DNA sequences. The X-ray intensity data set was collected on ETS–cfos promoter DNA complex crystal at 3.1 Å resolution to analyze the structure by molecular replacement technique. The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS–cfos DNA complex crystal at a wavelength of 0.9725 Å on the BM14 synchrotron beamline at the ESRF, France. The ETS–cfos DNA complex crystal belonged to space group C222 1 , with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS–cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein and cfos DNA as search models. Structure analysis of the ETS–cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55

  10. Crystallization and preliminary X-ray diffraction analysis of the carbohydrate-recognizing domain (VP8*) of bovine rotavirus strain NCDV

    International Nuclear Information System (INIS)

    Yu, Xing; Guillon, Annabel; Szyczew, Alex J.; Kiefel, Milton J.; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2008-01-01

    NCDV VP8* 64–224 was expressed in E. coli, purified and crystallized in the presence of a sialic acid derivative. X-ray diffraction data were obtained to a resolution of 2.0 Å and the crystallographic structure was determined by molecular replacement. The infectivity of rotavirus is dramatically enhanced by proteolytic cleavage of its outer layer VP4 spike protein into two functional domains, VP8* and VP5*. The carbohydrate-recognizing domain VP8* is proposed to bind sialic acid-containing host cell-surface glycans and this is followed by a series of subsequent virus–cell interactions. Live attenuated human and bovine rotavirus vaccine candidates for the prevention of gastroenteritis have been derived from bovine rotavirus strain NCDV. The NCDV VP8* 64–224 was overexpressed, purified to homogeneity and crystallized in the presence of an N-acetylneuraminic acid derivative. X-ray diffraction data were collected to a resolution of 2.0 Å and the crystallographic structure of NCDV VP8* 64–224 was determined by molecular replacement

  11. Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan.

    Science.gov (United States)

    Vieira, Diana; Figueiredo, Teresa A; Verma, Anil; Sobral, Rita G; Ludovice, Ana M; de Lencastre, Hermínia; Trincao, Jose

    2014-05-01

    Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

  12. Crystallization and preliminary X-ray analysis of a U2AF65 variant in complex with a polypyrimidine-tract analogue by use of protein engineering

    International Nuclear Information System (INIS)

    Sickmier, E. Allen; Frato, Katherine E.; Kielkopf, Clara L.

    2006-01-01

    A complex of the essential splicing factor U2AF 65 and a deoxyuridine oligonucleotide has been crystallized by modification of an interdomain linker. The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF 65 ) binds the polypyrimidine tract near the 3′ splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF 65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein–nucleic acid complex. Cocrystals of a U2AF 65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 Å resolution and contain one complex per asymmetric unit

  13. Crystallization, preliminary X-ray diffraction and structure solution of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti L5-30

    International Nuclear Information System (INIS)

    Leduc, Yvonne A.; Phenix, Christopher P.; Puttick, Jennifer; Nienaber, Kurt; Palmer, David R. J.; Delbaere, Louis T. J.

    2005-01-01

    MosA from S. meliloti L5-30 has been crystallized in solution with pyruvate and the 2.3 Å resolution structure has been solved by molecular replacement using E. coli dihydrodipicolinate synthase as the model. The structure of MosA, a dihydrodipicolinate synthase and reported methyltransferase from Sinorhizobium meliloti, has been solved using molecular replacement with Escherichia coli dihydrodipicolinate synthase as the model. A crystal grown in the presence of pyruvate diffracted X-rays to 2.3 Å resolution using synchrotron radiation and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 69.14, b = 138.87, c = 124.13 Å

  14. Purification, crystallization and preliminary X-ray diffraction analysis of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

    International Nuclear Information System (INIS)

    Huculeci, Radu; Buts, Lieven; Lenaerts, Tom; Nuland, Nico A. J. van; Garcia-Pino, Abel

    2012-01-01

    The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide

  15. Crystallization and preliminary X-ray analysis of a C-terminal fragment of FlgJ, a putative flagellar rod cap protein from Salmonella

    International Nuclear Information System (INIS)

    Kikuchi, Yuki; Matsunami, Hideyuki; Yamane, Midori; Imada, Katsumi; Namba, Keiichi

    2008-01-01

    A C-terminal fragment of Salmonella FlgJ, FlgJ 120–316 , which has peptidoglycan-hydrolysing activity, has been overproduced, purified and crystallized and the crystals have been characterized by X-ray diffraction. The formation of the bacterial flagellar axial structure, including the filament, the hook and the rod, requires the attachment of a cap complex to the distal end of the growing structure. Because the rod penetrates the peptidoglycan (PG) layer, the rod cap complex is thought to have PG-hydrolyzing activity. FlgJ is a putative rod cap protein whose C-terminal region shows sequence similarity to known muramidases. In this study, FlgJ 120–316 , a C-terminal fragment of FlgJ which contains the muramidase region, was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 3350 as a crystallizing agent and belonged to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.8, b = 43.9, c = 108.5 Å. Anomalous difference Patterson maps calculated from the diffraction data set of a selenomethionine-labelled crystal showed significant peaks in the Harker sections, indicating that the data were suitable for structure determination

  16. Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

    Energy Technology Data Exchange (ETDEWEB)

    Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki [National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba-shi, Ibaraki 305-8566 (Japan)

    2013-04-30

    The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å{sup 3} Da{sup −1} and a solvent content of 59%.

  17. Crystallization and preliminary X-ray data collection of the Escherichia coli lipoproteins BamC, BamD and BamE

    International Nuclear Information System (INIS)

    Albrecht, Reinhard; Zeth, Kornelius

    2010-01-01

    The cloning, purification and crystallization of the E. coli lipoproteins BamC, BamD and BamE is reported. X-ray diffraction data at high resolution were obtained for each of the proteins or protein domains. In Escherichia coli, the β-barrel assembly machinery (or BAM complex) mediates the recognition, insertion and assembly of outer membrane proteins. The complex consists of the integral membrane protein BamA (an Omp85-family member) and the lipoproteins BamB, BamC, BamD and BamE. The purification and crystallization of BamC, BamD and BamE, each lacking the N-terminal membrane anchor, is described. While the smallest protein BamE yielded crystals under conventional conditions, BamD only crystallized after stabilization with urea. Full-length BamC did not crystallize, but was cleaved by subtilisin into two domains which were subsequently crystallized independently. High-resolution data were acquired from all proteins

  18. Crystallization and preliminary X-ray diffraction analysis of the Cmr2–Cmr3 subcomplex in the CRISPR–Cas RNA-silencing effector complex

    International Nuclear Information System (INIS)

    Osawa, Takuo; Inanaga, Hideko; Numata, Tomoyuki

    2013-01-01

    The Cmr2–Cmr3 subcomplex from P. furiosus was co-crystallized with 3′-AMP. X-ray diffraction data for the crystals were collected to 2.6 Å resolution using a synchrotron-radiation source. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, found in prokaryotes, are transcribed to produce CRISPR RNAs (crRNAs). The Cmr proteins (Cmr1–6) and crRNA form a ribonucleoprotein complex that degrades target RNAs derived from invading genetic elements. Cmr2dHD, a Cmr2 variant lacking the N-terminal putative HD nuclease domain, and Cmr3 were co-expressed in Escherichia coli cells and co-purified as a complex. The Cmr2dHD–Cmr3 complex was co-crystallized with 3′-AMP by the vapour-diffusion method. The crystals diffracted to 2.6 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group I222, with unit-cell parameters a = 103.9, b = 136.7, c = 192.0 Å. The asymmetric unit of the crystals is expected to contain one Cmr2dHD–Cmr3 complex with a Matthews coefficient of 3.0 Å 3 Da −1 and a solvent content of 59%

  19. Complex assembly, crystallization and preliminary X-ray crystallographic studies of rhesus macaque MHC Mamu-A*01 complexed with an immunodominant SIV-Gag nonapeptide

    International Nuclear Information System (INIS)

    Chu, Fuliang; Lou, Zhiyong; Gao, Bin; Bell, John I.; Rao, Zihe; Gao, George F.

    2005-01-01

    Crystallization of the first rhesus macaque MHC class I complex. Simian immunodeficiency virus (SIV) infection in rhesus macaques has been used as the best model for the study of human immunodeficiency virus (HIV) infection in humans, especially in the cytotoxic T-lymphocyte (CTL) response. However, the structure of rhesus macaque (or any other monkey model) major histocompatibility complex class I (MHC I) presenting a specific peptide (the ligand for CTL) has not yet been elucidated. Here, using in vitro refolding, the preparation of the complex of the rhesus macaque MHC I allele (Mamu-A*01) with human β 2 m and an immunodominant peptide, CTPYDINQM (Gag-CM9), derived from SIV Gag protein is reported. The complex (45 kDa) was crystallized; the crystal belongs to space group I422, with unit-cell parameters a = b = 183.8, c = 155.2 Å. The crystal contains two molecules in the asymmetric unit and diffracts X-rays to 2.8 Å resolution. The structure is being solved by molecular replacement and this is the first attempt to determined the crystal structure of a peptide–nonhuman primate MHC complex

  20. Crystallization and preliminary X-ray crystallographic analysis of highly thermostable L2 lipase from the newly isolated Bacillus sp. L2

    International Nuclear Information System (INIS)

    Shariff, Fairolniza Mohd; Rahman, Raja Noor Zaliha Raja Abd.; Ali, Mohd Shukuri Mohamad; Chor, Adam Leow Thean; Basri, Mahiran; Salleh, Abu Bakar

    2010-01-01

    Thermostable recombinant L2 lipase from thermophilic Bacillus sp. L2 has been crystallized by using counter-diffusion method and diffracted to 2.7 Å resolution. The crystal belongs to the primitive orthorhombic space group P2 1 2 1 2 1 with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. Purified thermostable recombinant L2 lipase from Bacillus sp. L2 was crystallized by the counter-diffusion method using 20% PEG 6000, 50 mM MES pH 6.5 and 50 mM NaCl as precipitant. X-ray diffraction data were collected to 2.7 Å resolution using an in-house Bruker X8 PROTEUM single-crystal diffractometer system. The crystal belonged to the primitive orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 87.44, b = 94.90, c = 126.46 Å. The asymmetric unit contained one single molecule of protein, with a Matthews coefficient (V M ) of 2.85 Å 3 Da −1 and a solvent content of 57%

  1. Expression, purification, crystallization and preliminary X-ray analysis of a novel N-substituted branched-chain l-amino-acid dioxygenase from Burkholderia ambifaria AMMD

    International Nuclear Information System (INIS)

    Qin, Hui-Min; Miyakawa, Takuya; Nakamura, Akira; Xue, You-Lin; Kawashima, Takashi; Kasahara, Takuya; Hibi, Makoto; Ogawa, Jun; Tanokura, Masaru

    2012-01-01

    Diffraction data were collected to a limiting resolution of 2.4 Å from a crystal of selenomethionyl-labelled SadA, an l-amino-acid dioxygenase. Ferrous ion- and α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA) catalyzes the C3-hydroxylation of N-substituted branched-chain l-amino acids, especially N-succinyl-l-leucine, coupled to the conversion of α-ketoglutarate to succinate and CO 2 . SadA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted SadA were obtained using a reservoir solution containing PEG 3000 as the precipitant at pH 9.5 and diffracted X-rays to 2.4 Å resolution. The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.3, b = 70.9, c = 148.2 Å. The calculated Matthews coefficient (V M = 2.1 Å 3 Da −1 , 41% solvent content) suggested that the crystal contains two molecules per asymmetric unit

  2. X-ray conductivity of ZnSe single crystals

    Energy Technology Data Exchange (ETDEWEB)

    Degoda, V. Ya., E-mail: degoda@univ.kiev.ua; Podust, G. P. [Taras Shevchenko Kyiv National University, Physics Department (Ukraine)

    2016-05-15

    The experimental I–V and current–illuminance characteristics of the X-ray conductivity and X-ray luminescence of zinc-selenide single crystals feature a nonlinear shape. The performed theoretical analysis of the kinetics of the X-ray conductivity shows that even with the presence of shallow and deep traps for free charge carriers in a semiconductor sample, the integral characteristics of the X-ray conductivity (the current–illuminance and I–V dependences) should be linear. It is possible to assume that the nonlinearity experimentally obtained in the I–V and current–illuminance characteristics can be caused by features of the generation of free charge carriers upon X-ray irradiation, i.e., the generation of hundreds of thousands of free charge carriers of opposite sign in a local region with a diameter of <1 μm and Coulomb interaction between the free charge carriers of opposite signs.

  3. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    International Nuclear Information System (INIS)

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-01-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2 1 and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2 1 , with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å 3 Da −1 and a solvent content of 50%

  4. Crystallization and preliminary X-ray diffraction studies of BmooPLA2-I, a platelet-aggregation inhibitor and hypotensive phospholipase A2 from Bothrops moojeni venom

    International Nuclear Information System (INIS)

    Salvador, Guilherme H. M.; Marchi-Salvador, Daniela P.; Silveira, Lucas B.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    BmooPLA 2 -I, an acidic, catalytic and nontoxic phospholipase A 2 from B. moojeni venom that is able to inhibit platelet aggregation and induce a hypotensive effect, has been crystallized. An X-ray diffraction data set was collected to 1.6 Å resolution and a molecular-replacement solution was obtained. Phospholipases A 2 (PLA 2 s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA 2 s. BmooPLA 2 -I is an acidic, nontoxic and catalytic PLA 2 isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA 2 -I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C222 1 , with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA 2 -I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA 2 from B. jararacussu (BthA-I) and other toxic and nontoxic PLA 2 s may provide important insights into the functional aspects of this class of proteins

  5. X-ray characterization of curved crystals for hard x-ray astronomy

    Science.gov (United States)

    Buffagni, Elisa; Bonnini, Elisa; Ferrari, Claudio; Virgilli, Enrico; Frontera, Filippo

    2015-05-01

    Among the methods to focus photons the diffraction in crystals results as one of the most effective for high energy photons. An assembling of properly oriented crystals can form a lens able to focus x-rays at high energy via Laue diffraction in transmission geometry; this is a Laue lens. The x-ray diffraction theory provides that the maximum diffraction efficiency is achieved in ideal mosaic crystals, but real mosaic crystals show diffraction efficiencies several times lower than the ideal case due to technological problems. An alternative and convenient approach is the use of curved crystals. We have recently optimized an efficient method based on the surface damage of crystals to produce self-standing uniformly curved Si, GaAs and Ge tiles of thickness up to 2-3 mm and curvature radii R down to a few meters. We show that, for curved diffracting planes, such crystals have a diffraction efficiency nearly forty times higher than the diffraction efficiency of perfect similar flat crystals, thus very close to that of ideal mosaic crystals. Moreover, in an alternative configuration where the diffracting planes are perpendicular to the curved ones, a focusing effect occurs and will be shown. These results were obtained for several energies between 17 and 120 keV with lab sources or at high energy facilities such as LARIX at Ferrara (Italy), ESRF at Grenoble (France), and ANKA at Karlsruhe (Germany).

  6. Crystallization and preliminary X-ray analysis of the complexes between a Fab and two forms of human insulin-like growth factor II

    International Nuclear Information System (INIS)

    Newman, Janet; Cohen, Edward H.; Cosgrove, Leah; Kopacz, Kris; Dransfield, Daniel T.; Adams, Timothy E.; Peat, Thomas S.

    2009-01-01

    Complexes of both hIGF-II and hIGF-IIE with a Fab have been crystallized and investigated by X-ray analysis. Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab–IGF-II complexes (M64-F02–IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 Å resolution and belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 Å. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 50.7, b = 107, c = 111.5 Å, and also diffracted X-rays to 2.2 Å resolution

  7. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the central zinc-binding domain of the human Mcm10 DNA-replication factor

    International Nuclear Information System (INIS)

    Jung, Nam Young; Bae, Won Jin; Chang, Jeong Ho; Kim, Young Chang; Cho, Yunje

    2008-01-01

    Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 Å on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 Å. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit

  8. Crystallization and preliminary X-ray analysis of a truncated mutant of yeast nuclear thiol peroxidase, a novel atypical 2-Cys peroxiredoxin

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jongkeun; Choi, Soonwoong [Department of Chemistry, Seoul National University, Seoul 151-742 (Korea, Republic of); Choi, Jungwon [Department of Chemistry, The University of Suwon, Suwon 445-743 (Korea, Republic of); Cha, Mee-Kyung; Kim, Il-Han [Department of Biochemistry, Paichai University, Taejon 302-735 (Korea, Republic of); Shin, Whanchul, E-mail: nswcshin@plaza.snu.ac.kr [Department of Chemistry, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2005-07-01

    A double mutant of yeast nuclear thiol peroxidase has been crystallized in a truncated form. The crystal belongs to space group P3{sub 2}, with unit-cell parameters a = b = 37.54, c = 83.26 Å. A diffraction data set has been collected to 1.8 Å resolution. Saccharomyces cerevisiae nTPx is a thioredoxin-dependent thiol peroxidase that is localized in the nucleus. nTPx belongs to the C-type atypical 2-Cys peroxiredoxin family members, which are frequently called BCPs or PrxQs. A double mutant (C107S/C112S) of nTPx overexpressed in Escherichia coli was spontaneously degraded upon freezing and thawing and its truncated form (residues 57–215; MW = 17837 Da) was crystallized with PEG 3350 and mercury(II) acetate as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.8 Å resolution using X-ray synchrotron radiation. The crystals belong to the trigonal space group P3{sub 2}, with unit-cell parameters a = b = 37.54, c = 83.26 Å. The asymmetric unit contains one molecule of truncated mutant nTPx, with a corresponding V{sub M} of 1.91 Å{sup 3} Da{sup −1} and a solvent content of 35.5%.

  9. Crystallization and preliminary X-ray analysis of a truncated mutant of yeast nuclear thiol peroxidase, a novel atypical 2-Cys peroxiredoxin

    International Nuclear Information System (INIS)

    Choi, Jongkeun; Choi, Soonwoong; Choi, Jungwon; Cha, Mee-Kyung; Kim, Il-Han; Shin, Whanchul

    2005-01-01

    A double mutant of yeast nuclear thiol peroxidase has been crystallized in a truncated form. The crystal belongs to space group P3 2 , with unit-cell parameters a = b = 37.54, c = 83.26 Å. A diffraction data set has been collected to 1.8 Å resolution. Saccharomyces cerevisiae nTPx is a thioredoxin-dependent thiol peroxidase that is localized in the nucleus. nTPx belongs to the C-type atypical 2-Cys peroxiredoxin family members, which are frequently called BCPs or PrxQs. A double mutant (C107S/C112S) of nTPx overexpressed in Escherichia coli was spontaneously degraded upon freezing and thawing and its truncated form (residues 57–215; MW = 17837 Da) was crystallized with PEG 3350 and mercury(II) acetate as precipitants using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.8 Å resolution using X-ray synchrotron radiation. The crystals belong to the trigonal space group P3 2 , with unit-cell parameters a = b = 37.54, c = 83.26 Å. The asymmetric unit contains one molecule of truncated mutant nTPx, with a corresponding V M of 1.91 Å 3 Da −1 and a solvent content of 35.5%

  10. Crystallization and preliminary X-ray studies of TON-1713 from Thermococcus onnurineus NA1, a putative member of the haloacid dehalogenase superfamily

    International Nuclear Information System (INIS)

    Le, Binh Van; Lee, Hyun Sook; Cho, Yona; Kang, Sung Gyun; Kim, Dong Young; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2007-01-01

    A putative member of the haloacid dehalogenase superfamily from T. onnurineus has been expressed, purified and crystallized using 1.6 M magnesium sulfate as a precipitant. The crystals belonged to the triclinic space group P1 and diffracted to 1.8 Å resolution. The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON-1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°

  11. Crystallization and preliminary X-ray crystallographic study of the extracellular domain of the 4-1BB ligand, a member of the TNF family

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Jung-Sue; Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Ahn, Byungchan; Kwon, Byoung Se [Immunomodulation Research Center, Ulsan University, Ulsan 680-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2006-01-01

    The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. The 4-1BB ligand, a member of the tumour necrosis factor (TNF) family, is an important co-stimulatory molecule that plays a key role in the clonal expansion and survival of CD8+ T cells. Signalling through binding of the 4-1BB ligand and 4-1BB has been reported to enhance CD8+ T-cell expansion and protect activated CD8+ T cells from death. The 4-1BB ligand is an integral protein expressed on activated antigen-presenting cells. The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from these crystals to 2.8 Å resolution and the crystals belong to space group C2, with unit-cell parameters a = 114.6, b = 73.8, c = 118.50 Å, β = 115.5°.

  12. Crystallization and preliminary X-ray crystallographic study of the extracellular domain of the 4-1BB ligand, a member of the TNF family

    International Nuclear Information System (INIS)

    Byun, Jung-Sue; Kim, Dong-Uk; Ahn, Byungchan; Kwon, Byoung Se; Cho, Hyun-Soo

    2005-01-01

    The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. The 4-1BB ligand, a member of the tumour necrosis factor (TNF) family, is an important co-stimulatory molecule that plays a key role in the clonal expansion and survival of CD8+ T cells. Signalling through binding of the 4-1BB ligand and 4-1BB has been reported to enhance CD8+ T-cell expansion and protect activated CD8+ T cells from death. The 4-1BB ligand is an integral protein expressed on activated antigen-presenting cells. The extracellular domain of the 4-1BB ligand fused with glutathione-S-transferase was expressed in Escherichia coli (Origami) and purified by using affinity and ion-exchange column chromatographic methods. Crystals of the 4-1BB ligand were obtained at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from these crystals to 2.8 Å resolution and the crystals belong to space group C2, with unit-cell parameters a = 114.6, b = 73.8, c = 118.50 Å, β = 115.5°

  13. Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core from Nostoc punctiforme

    International Nuclear Information System (INIS)

    Lee, Jun Hyuck; Park, Soo Jeong; Rho, Seong-Hwan; Im, Young Jun; Kim, Mun-Kyoung; Kang, Gil Bu; Eom, Soo Hyun

    2005-01-01

    The GluR0 ligand-binding core from N. punctiforme was expressed, purified and crystallized in the presence of l-glutamate. A diffraction data set was collected to a resolution of 2.1 Å. GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The l-glutamate-complexed crystal belongs to space group C222 1 , with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 Å. The crystals contain three subunits in the asymmetric unit, with a V M value of 2.49 Å 3 Da −1 . The diffraction limit of the l-glutamate complex data set was 2.1 Å using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea)

  14. Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core from Nostoc punctiforme

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jun Hyuck; Park, Soo Jeong; Rho, Seong-Hwan; Im, Young Jun; Kim, Mun-Kyoung; Kang, Gil Bu; Eom, Soo Hyun, E-mail: eom@gist.ac.kr [Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2005-11-01

    The GluR0 ligand-binding core from N. punctiforme was expressed, purified and crystallized in the presence of l-glutamate. A diffraction data set was collected to a resolution of 2.1 Å. GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The l-glutamate-complexed crystal belongs to space group C222{sub 1}, with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 Å. The crystals contain three subunits in the asymmetric unit, with a V{sub M} value of 2.49 Å{sup 3} Da{sup −1}. The diffraction limit of the l-glutamate complex data set was 2.1 Å using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea)

  15. Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD)

    International Nuclear Information System (INIS)

    Pandey, Nishant Kumar; Pal, Ravi Kant; Kashyap, Maruthi; Bhavesh, Neel Sarovar

    2012-01-01

    The Escherichia coli PapD-like protein (EcpD), from uropathogenic Escherichia coli (UPEC), which is a periplasmic chaperon of Yad fimbriae was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.67 Å resolution and belonged to space group C222 1 . Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å 3 Da −1 , with 59% solvent content. Initial phases were determined by molecular replacement

  16. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Fulton, Zara [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Crellin, Paul K.; Brammananth, Rajini [Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Zaker-Tabrizi, Leyla [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Coppel, Ross L. [Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Rossjohn, Jamie, E-mail: jamie.rossjohn@med.monash.edu.au; Beddoe, Travis, E-mail: jamie.rossjohn@med.monash.edu.au [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia)

    2008-05-01

    MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.

  17. Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crab Cancer pagurus

    International Nuclear Information System (INIS)

    Hehemann, Jan-Hendrik; Redecke, Lars; Perbandt, Markus; Saborowski, Reinhard; Betzel, Christian

    2007-01-01

    Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution. The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 Å resolution, respectively. The crystal of C.p.TryII belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 Å. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P2 1 3 with unit-cell parameters a = b = c = 215.4 Å, are assumed to contain 12 molecules per asymmetric unit

  18. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    International Nuclear Information System (INIS)

    Zhang, Yang-De; Li, Hao; Liu, Hui; Pan, Yi-Feng

    2007-01-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2 1 2 1 2 1 and tetragonal P4 1 2 1 2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8* 65–224 structure was determined by molecular replacement

  19. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yang-De, E-mail: zhangyd1960@yahoo.com.cn; Li, Hao [National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China); Liu, Hui; Pan, Yi-Feng [Biochemistry Laboratory, Institution of Biomedical Engineering, Central South University, Hunan Province (China); National Hepatobiliary and Enteric Surgery Research Center of The Ministry of Health, Xiangya Hospital, Central South University, Hunan Province (China)

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  20. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    International Nuclear Information System (INIS)

    Fulton, Zara; Crellin, Paul K.; Brammananth, Rajini; Zaker-Tabrizi, Leyla; Coppel, Ross L.; Rossjohn, Jamie; Beddoe, Travis

    2008-01-01

    MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4 1 2 1 2

  1. Crystallization and preliminary X-ray crystallographic studies of Rab6A′(Q72L): a GTP-locked form

    International Nuclear Information System (INIS)

    Shin, Young-Cheul; Jang, Tae-Ho; Yoon, Jong Hwan; Jeon, Ju-Hong; Park, Hyun Ho

    2012-01-01

    Rab6A′(Q72L), a constitutively active GTP-binding form of Rab6A, was purified and crystallized. The crystals were found to belong to space group P22 1 2 1 , with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The crystals were obtained at 293 K and diffracted to a resolution of 1.9 Å. Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A′. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A′ and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A′(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A′(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22 1 2 1 with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules

  2. Expression, purification, crystallization and preliminary X-ray analysis of eCGP123, an extremely stable monomeric green fluorescent protein with reversible photoswitching properties

    International Nuclear Information System (INIS)

    Don Paul, Craig; Traore, Daouda A. K.; Byres, Emma; Rossjohn, Jamie; Devenish, Rodney J.; Kiss, Csaba; Bradbury, Andrew; Wilce, Matthew C. J.; Prescott, Mark

    2011-01-01

    eCGP123, an extremely stable GFP with photoswitching properties, has been expressed, purified and crystallized. A diffraction data set has been collected at 2.10 Å resolution. Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, β = 89.92, γ = 104.03°. The Matthews coefficient (V M = 2.26 Å 3 Da −1 ) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules

  3. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of SET/TAF-Iβ ΔN from Homo sapiens

    International Nuclear Information System (INIS)

    Xu, Zhen; Yang, Weili; Shi, Nuo; Gao, Yongxiang; Teng, Maikun; Niu, Liwen

    2010-01-01

    The SET/TAF-Iβ that lacked the first 22 residues of the N-terminus from Homo sapiens was recombinantly expressed in Escherichia coli and crystallized. X-ray diffraction data were collected to 2.7 Å resolution. The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iβ (TAF-Iβ), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-Iβ ΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His 6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 Å resolution and belonged to space group P4 3 2 1 2

  4. Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB-G0286605 protein from the social amoeba Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Kim, Min-Kyu; Yim, Hyung-Soon; Kang, Sa-Ouk

    2010-01-01

    In order to investigate its structure and function, the NmrA-like domain-containing DDB-G0286605 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.64 Å. The DDB-G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Å resolution data set was collected using synchrotron radiation. The DDB-G0286605 protein crystals belonged to space group P2 1 , with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB-G0286605 protein and the search models (DDB-G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively)

  5. Crystallization and preliminary X-ray crystallographic analysis of BxlA, an intracellular β-d-xylosidase from Streptomyces thermoviolaceus OPC-520

    International Nuclear Information System (INIS)

    Morioka, Hideaki; Miki, Yasuhiro; Saito, Kei; Tomoo, Koji; Ishida, Toshimasa; Hasegawa, Tomokazu; Yamano, Akihito; Takada, Chiaki; Miyamoto, Katsushiro; Tsujibo, Hiroshi

    2010-01-01

    BxlA from Streptomyces thermoviolaceus OPC-520 (molecular weight 82 kDa) was crystallized by the hanging-drop vapour-diffusion method at 289 K. BxlA from Streptomyces thermoviolaceus OPC-520, together with the extracellular BxlE and the integral membrane proteins BxlF and BxlG, constitutes a xylanolytic system that participates in the intracellular transport of xylan-degradation products and the production of xylose. To elucidate the mechanism of the hydrolytic degradation of xylooligosaccharides to xylose at the atomic level, X-ray structural analysis of BxlA was attempted. The recombinant BxlA protein (molecular weight 82 kDa) was crystallized by the hanging-drop vapour-diffusion method at 289 K. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 142.2, b = 129.5, c = 101.4 Å, β = 119.8°, and contained two molecules per asymmetric unit (V M = 2.47 Å 3 Da −1 ). Diffraction data were collected to a resolution to 2.50 Å and provided a data set with an overall R merge of 8.3%

  6. Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta)

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, Andrew A., E-mail: andrewmc@embl.fr [European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France); Biget, Laurent [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); Lin, Chenwei [Department of Plant Breeding and Genetics, Department of Plant Biology, Cornell University, Ithaca, NY 14853 (United States); Petiard, Vincent [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); Tanksley, Steve D. [Department of Plant Breeding and Genetics, Department of Plant Biology, Cornell University, Ithaca, NY 14853 (United States); McCarthy, James G. [Nestlé Research and Development, 101 Avenue Gustave Eiffel, Notre-Dame D’Oe, 37097 Tours (France); European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, 38042 Grenoble (France)

    2007-04-01

    The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2{sub 1}2{sub 1}2{sub 1} for XMT and C222{sub 1} for DXMT. X-ray diffraction to 2.8 Å for XMT and to 2.5 Å for DXMT have been collected on beamline ID23-1 at the ESRF.

  7. Cloning, expression, crystallization and preliminary X-ray analysis of the XMT and DXMT N-methyltransferases from Coffea canephora (robusta)

    International Nuclear Information System (INIS)

    McCarthy, Andrew A.; Biget, Laurent; Lin, Chenwei; Petiard, Vincent; Tanksley, Steve D.; McCarthy, James G.

    2007-01-01

    The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). Caffeine is a secondary metabolite produced by a variety of plants including Coffea canephora (robusta) and there is growing evidence that caffeine is part of a chemical defence strategy protecting young leaves and seeds from potential predators. The genes encoding XMT and DXMT, the enzymes from Coffea canephora (robusta) that catalyse the three independent N-methyl transfer reactions in the caffeine-biosynthesis pathway, have been cloned and the proteins have been expressed in Escherichia coli. Both proteins have been crystallized in the presence of the demethylated cofactor S-adenosyl-l-cysteine (SAH) and substrate (xanthosine for XMT and theobromine for DXMT). The crystals are orthorhombic, with space group P2 1 2 1 2 1 for XMT and C222 1 for DXMT. X-ray diffraction to 2.8 Å for XMT and to 2.5 Å for DXMT have been collected on beamline ID23-1 at the ESRF

  8. Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein

    International Nuclear Information System (INIS)

    Petty, Tom J.; Nishimura, Taisuke; Emamzadah, Soheila; Gabus, Caroline; Paszkowski, Jerzy; Halazonetis, Thanos D.; Thore, Stéphane

    2010-01-01

    In order to investigate its function in transcriptional gene silencing, the highly conserved motif 2 from A. thaliana Morpheus’ molecule 1 protein was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 3.2 Å. Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing

  9. Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Noboru; Kumarevel, Thirumananseri, E-mail: tskvel@spring8.or.jp; Matsunaga, Emiko; Shinkai, Akeo [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Kuramitsu, Seiki [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Department of Biological Sciences, Graduate School of Science, Osaka University, Tayonaka, Osaka 560-0043 (Japan); Yokoyama, Shigeyuki, E-mail: tskvel@spring8.or.jp [RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan); Genomic Sciences Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2007-11-01

    A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way. The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 Å and a = b = 103.846, c = 73.992 Å, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

  10. Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

    International Nuclear Information System (INIS)

    Nakano, Noboru; Kumarevel, Thirumananseri; Matsunaga, Emiko; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki

    2007-01-01

    A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way. The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 Å and a = b = 103.846, c = 73.992 Å, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit