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Sample records for crystal x-ray analysis

  1. Crystal quality analysis and improvement using x-ray topography

    International Nuclear Information System (INIS)

    Maj, J.; Goetze, K.; Macrander, A.; Zhong, Y.; Huang, X.; Maj, L.

    2008-01-01

    The Topography X-ray Laboratory of the Advanced Photon Source (APS) at Argonne National Laboratory operates as a collaborative effort with APS users to produce high performance crystals for APS X-ray beamline experiments. For many years the topography laboratory has worked closely with an on-site optics shop to help ensure the production of crystals with the highest quality, most stress-free surface finish possible. It has been instrumental in evaluating and refining methods used to produce high quality crystals. Topographical analysis has shown to be an effective method to quantify and determine the distribution of stresses, to help identify methods that would mitigate the stresses and improve the Rocking curve, and to create CCD images of the crystal. This paper describes the topography process and offers methods for reducing crystal stresses in order to substantially improve the crystal optics.

  2. Parallelization for X-ray crystal structural analysis program

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Hiroshi [Japan Atomic Energy Research Inst., Tokyo (Japan); Minami, Masayuki; Yamamoto, Akiji

    1997-10-01

    In this report we study vectorization and parallelization for X-ray crystal structural analysis program. The target machine is NEC SX-4 which is a distributed/shared memory type vector parallel supercomputer. X-ray crystal structural analysis is surveyed, and a new multi-dimensional discrete Fourier transform method is proposed. The new method is designed to have a very long vector length, so that it enables to obtain the 12.0 times higher performance result that the original code. Besides the above-mentioned vectorization, the parallelization by micro-task functions on SX-4 reaches 13.7 times acceleration in the part of multi-dimensional discrete Fourier transform with 14 CPUs, and 3.0 times acceleration in the whole program. Totally 35.9 times acceleration to the original 1CPU scalar version is achieved with vectorization and parallelization on SX-4. (author)

  3. Rapid X-ray crystal structure analysis in few second measurements using microstrip gas chamber

    CERN Document Server

    Ochi, A; Tanimori, T; Ohashi, Y; Toyokawa, H; Nishi, Y; Nishi, Y; Nagayoshi, T; Koishi, S

    2001-01-01

    X-ray crystal structure analysis using microstrip gas chamber was successfully carried out in a measurement time within a few seconds. The continuous rotation photograph method, in which most of the diffraction peaks can be obtained within one continuous rotation of the sample crystal (without stopping or oscillation), was applied for this measurement. As an example, the structure of a single crystal of ammonium bitartrate (r=1 mm, spherical) was measured. Diffraction spots from the sample, which were sufficient to obtain crystal structure, were successfully obtained by taking only 2 s measurements with a commercially available laboratory X-ray source.

  4. Quantitative analysis of thermal diffuse X-ray scattering on single crystals. Communication 2. FCC metals

    International Nuclear Information System (INIS)

    Najsh, V.E.; Novoselova, T.V.; Sagaradze, I.V.; Kvyatkovskij, B.E.; Fedorov, V.I.; Chernenkov, Yu.P.

    1994-01-01

    With the use of X-ray diffractometer a study was made into the intensity of diffuse scattering in Ni crystals with FCC lattice. Earlier accomplished quantitative analysis for BCC crystals was extended to FCC lattices. Comparative evaluation was made for cooperative thermal oscillation patterns and corresponding diffuse scattering in crystals of various structures. Measurements on FCC crystals were carried out at room temperature using AgK a lpha-radiation in 96 points of Ni crystal. 8 refs., 4 figs

  5. 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.

  6. 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

  7. Advanced x-ray stress analysis method for a single crystal using different diffraction plane families

    International Nuclear Information System (INIS)

    Imafuku, Muneyuki; Suzuki, Hiroshi; Sueyoshi, Kazuyuki; Akita, Koichi; Ohya, Shin-ichi

    2008-01-01

    Generalized formula of the x-ray stress analysis for a single crystal with unknown stress-free lattice parameter was proposed. This method enables us to evaluate the plane stress states with any combination of diffraction planes. We can choose and combine the appropriate x-ray sources and diffraction plane families, depending on the sample orientation and the apparatus, whenever diffraction condition is satisfied. The analysis of plane stress distributions in an iron single crystal was demonstrated combining with the diffraction data for Fe{211} and Fe{310} plane families

  8. Synthesis and single crystal X-ray analysis of two griseofulvin metabolites

    DEFF Research Database (Denmark)

    Rønnest, Mads Holger; Harris, Pernille; Gotfredsen, Charlotte Held

    2010-01-01

    The two phenols, 6-O-desmethyl griseofulvin and 4-O-desmethyl griseofulvin are metabolites of the antifungal drug griseofulvin. Herein, we present an improved synthesis of the 6-phenol derivative, and an unequivocal proof of both structures by single-crystal X-ray analysis.......The two phenols, 6-O-desmethyl griseofulvin and 4-O-desmethyl griseofulvin are metabolites of the antifungal drug griseofulvin. Herein, we present an improved synthesis of the 6-phenol derivative, and an unequivocal proof of both structures by single-crystal X-ray analysis....

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Development of high-performance X-ray transparent crystallization plates for in situ protein crystal screening and analysis

    Energy Technology Data Exchange (ETDEWEB)

    Soliman, Ahmed S. M.; Warkentin, Matthew [Cornell University, Ithaca, New York (United States); Apker, Benjamin [MiTeGen LLC, Ithaca, New York (United States); Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, New York (United States); MiTeGen LLC, Ithaca, New York (United States)

    2011-07-01

    An optically, UV and X-ray transparent crystallization plate suitable for in situ analysis has been developed. The plate uses contact line pinning rather than wells to confine the liquids. X-ray transparent crystallization plates based upon a novel drop-pinning technology provide a flexible, simple and inexpensive approach to protein crystallization and screening. The plates consist of open cells sealed top and bottom by thin optically, UV and X-ray transparent films. The plates do not need wells or depressions to contain liquids. Instead, protein drops and reservoir solution are held in place by rings with micrometre dimensions that are patterned onto the bottom film. These rings strongly pin the liquid contact lines, thereby improving drop shape and position uniformity, and thus crystallization reproducibility, and simplifying automated image analysis of drop contents. The same rings effectively pin solutions containing salts, proteins, cryoprotectants, oils, alcohols and detergents. Strong pinning by rings allows the plates to be rotated without liquid mixing to 90° for X-ray data collection or to be inverted for hanging-drop crystallization. The plates have the standard SBS format and are compatible with standard liquid-handling robots.

  15. 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

  16. 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

  17. Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Minglian; Li, Zhenguo; Zheng, Wei; Lou, Zhiyong [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Chen, Guo-Qiang, E-mail: chengq@stu.edu.cn [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong (China)

    2006-08-01

    The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

  18. 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

  19. 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%

  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. Recombinant ACHT1 from Arabidopsis thaliana: crystallization and X-ray crystallographic analysis.

    Science.gov (United States)

    Pan, Weimin; Wang, Junchao; Yang, Ye; Liu, Lin; Zhang, Min

    2017-07-01

    Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7 Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space group C222 1 , with unit-cell parameters a = 102.7, b = 100.6, c = 92.8 Å.

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Data reduction and analysis for the graphite crystal X-ray spectrometer and polarimeter experiment flown aboard OSO-8 spacecraft

    Science.gov (United States)

    Novick, R.

    1980-01-01

    The documentation and software programs developed for the reception, initial processing (quickbook), and production analysis of data obtained by solar X-ray spectroscopy, stellar spectroscopy, and X-ray polarimetry experiments on OSO-8 are listed. The effectiveness and sensitivity of the Bragg crystal scattering instruments used are assessed. The polarization data polarimetric data obtained shows that some X-ray sources are polarized and that a larger polarimeter of this type is required to perform the measurements necessary to fully understand the physics of X-ray sources. The scanning Bragg crystal spectrometer was ideally suited for studying rapidly changing solar conditions. Observations of the Crab Nebula and pulsar, Cyg X-1, Cyg X-2, Cyg X-3, Sco X-1, Cen X-3, and Her X-1 are discussed as well as of 4U1656-53 and 4U1820-30. Evidence was obtained for iron line emission from Cyg X-3.

  8. 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

  9. 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°

  10. Double crystal X-ray analysis of phosphorus precipitation in supersaturated Si-P solid solutions

    International Nuclear Information System (INIS)

    Servidori, M.; Zini, Q.; Dal Monte, C.

    1983-01-01

    The physical nature of the electrically inactive phosphorus in silicon is investigated by double crystal X-ray diffraction measurements. This analysis is performed on laser annealed supersaturated samples, doped by ion implantation up to 5 x 10 21 cm -3 . After isothermal heat treatments, these solid solutions show marked reductions in the electrically active phosphorus concentration. In particular, 850 0 C heatings give rise to a carrier concentration which corresponds to the phosphorus solubility in equilibrium with the inactive dopant. This dopant is characterized by means of lattice strain measurements: they are found consistent with the presence of perfectly coherent cubic SiP precipitates. This result is in agreement with the one obtained in preceeding works by electrical measurements and transmission electron microscopy observations and contradicts the hypothesis that the excess dopant atoms are, at least in part, charged point defects (E-centres). (author)

  11. 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

  12. 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°

  13. Analysis and implementation of a space resolving spherical crystal spectrometer for x-ray Thomson scattering experiments.

    Science.gov (United States)

    Harding, E C; Ao, T; Bailey, J E; Loisel, G; Sinars, D B; Geissel, M; Rochau, G A; Smith, I C

    2015-04-01

    The application of a space-resolving spectrometer to X-ray Thomson Scattering (XRTS) experiments has the potential to advance the study of warm dense matter. This has motivated the design of a spherical crystal spectrometer, which is a doubly focusing geometry with an overall high sensitivity and the capability of providing high-resolution, space-resolved spectra. A detailed analysis of the image fluence and crystal throughput in this geometry is carried out and analytical estimates of these quantities are presented. This analysis informed the design of a new spectrometer intended for future XRTS experiments on the Z-machine. The new spectrometer collects 6 keV x-rays with a spherically bent Ge (422) crystal and focuses the collected x-rays onto the Rowland circle. The spectrometer was built and then tested with a foam target. The resulting high-quality spectra prove that a spherical spectrometer is a viable diagnostic for XRTS experiments.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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°

  2. single crystal growth, x-ray structure analysis, optical band gap

    African Journals Online (AJOL)

    2015-09-01

    Sep 1, 2015 ... Hg...Hgand Cl...Cl interactions are stabilizing the structures in 3D pattern. UV-vis absorption spectra illustrate the change in opticalband gap from 3.01eVto 3.42eV on replacing the metal halide group.Raman and Hyper-Raman tensors calculations were performed based on single crystal X-ray data and the ...

  3. Instrument and method for X-ray diffraction, fluorescence, and crystal texture analysis without sample preparation

    Science.gov (United States)

    Gendreau, Keith (Inventor); Martins, Jose Vanderlei (Inventor); Arzoumanian, Zaven (Inventor)

    2010-01-01

    An X-ray diffraction and X-ray fluorescence instrument for analyzing samples having no sample preparation includes a X-ray source configured to output a collimated X-ray beam comprising a continuum spectrum of X-rays to a predetermined coordinate and a photon-counting X-ray imaging spectrometer disposed to receive X-rays output from an unprepared sample disposed at the predetermined coordinate upon exposure of the unprepared sample to the collimated X-ray beam. The X-ray source and the photon-counting X-ray imaging spectrometer are arranged in a reflection geometry relative to the predetermined coordinate.

  4. 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

  5. Ultrasmall-angle X-ray scattering analysis of photonic crystal structure

    International Nuclear Information System (INIS)

    Abramova, V. V.; Sinitskii, A. S.; Grigor'eva, N. A.; Grigor'ev, S. V.; Belov, D. V.; Petukhov, A. V.; Mistonov, A. A.; Vasil'eva, A. V.; Tret'yakov, Yu. D.

    2009-01-01

    The results of an ultrasmall-angle X-ray scattering study of iron(III) oxide inverse opal thin films are presented. The photonic crystals examined are shown to have fcc structure with amount of stacking faults varying among the samples. The method used in this study makes it possible to easily distinguish between samples with predominantly twinned fcc structure and nearly perfect fcc stacking. The difference observed between samples fabricated under identical conditions is attributed to random layer stacking in the self-assembled colloidal crystals used as templates for fabricating the inverse opals. The present method provides a versatile tool for analyzing photonic crystal structure in studies of inverse opals made of various materials, colloidal crystals, and three-dimensional photonic crystals of other types.

  6. 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%.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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%

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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%

  20. 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%.

  1. 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.

  2. 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)

  3. 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

  4. 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

  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. Crystallization and X-ray analysis of the salmon-egg lectin SEL24K

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Kenji [Department of Animal Science, University of California, Davis 95616 (United States); Fisher, Andrew J. [Department of Chemistry, University of California, Davis 95616 (United States); Hedrick, Jerry L., E-mail: jlhedrick@ucdavis.edu [Department of Animal Science, University of California, Davis 95616 (United States)

    2007-05-01

    The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The 24 kDa egg lectin of Chinook salmon (Oncorhynchus tshawytscha) is released from the egg during the cortical reaction. The lectin functions in blocking polyspermy during the fertilization process. The egg lectin was purified by affinity chromatography from salmon eggs and crystallized by the hanging-drop vapor-diffusion method using 15/4 EO/OH (pentaerythritol ethoxylate) as a precipitant. The crystal diffracted synchrotron-radiation X-rays to 1.63 Å resolution. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 93.0, b = 73.6, c = 113.6 Å, α = 90, β = 92.82, γ = 90°. The crystal is likely to contain eight molecules in the asymmetric unit (V{sub M} = 2.3 Å{sup 3} Da{sup −1}), corresponding to a solvent content of 45.5%. A self-rotation function suggests an arrangement with 222 point symmetry within the asymmetric unit.

  7. 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

  8. 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

  9. 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

  10. 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)

  11. Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens

    International Nuclear Information System (INIS)

    Hou, Feng; Miyakawa, Takuya; Takeshita, Daijiro; Kataoka, Michihiko; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2012-01-01

    The purification and crystallization of 3-quinuclidinone reductase from A. tumefaciens allowed the collection of a diffraction data set to 1.72 Å resolution. (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2 1 , with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V M = 2.08 Å 3 Da −1 )

  12. 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}.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. Crystal structure and charge density analysis of Li2NH by synchrotron X-ray diffraction

    International Nuclear Information System (INIS)

    Noritake, T.; Nozaki, H.; Aoki, M.; Towata, S.; Kitahara, G.; Nakamori, Y.; Orimo, S.

    2005-01-01

    Complex hydrides, such as lithium amide (LiNH 2 ) and lithium imide (Li 2 NH), have recently been noticed as one of the most promising materials for reversible hydrogen storage. In this paper, we reveal the bonding nature of hydrogen in Li 2 NH crystal by synchrotron powder X-ray diffraction measurement at room temperature. The crystal structure was refined by Rietveld method and the charge density distribution was analyzed by maximum entropy method (MEM). The Li 2 NH crystal is anti-fluorite type structure (space group Fm3-bar m) consisting of Li and NH. Hydrogen atom occupies randomly the 48h (Wyckoff notation) sites around N atom. The refined lattice constant is a=5.0742(2)A. The charge density distribution around NH anion in Li 2 NH is almost spherical. The number of electrons within the sphere around the Li and NH is estimated from the obtained charge density distribution. As the result, the ionic charge is expressed as [Li 0.99+ ] 2 [NH] 1.21- . Therefore, it is confirmed experimentally that Li 2 NH is ionically bonded

  20. Auger and depth profile analysis of synthetic crystals for dispersion of soft x-rays

    International Nuclear Information System (INIS)

    Rachocki, K.D.; Brown, D.R.; Springer, R.W.; Arendt, P.N.

    1983-01-01

    Numerous samples have been fabricated and analyzed as part of a program to produce soft x-ray dispersion elements for various laboratory applications. The majority of this work has centered around the carbon/tungsten system, although several other low-Z/high-Z pairs have been investigated. This report describes the development of certain vacuum-deposition techniques for fabricating these dispersion elements, based upon results obtained from x-ray reflectivity measurements and Auger depth-profile analysis. The composition of the films is chiefly alternating layers of tungsten carbide and carbon. Excess carbon is introduced during the deposition of the tungsten to ensure that the carbide layer is fully stoichiometric. Layer thickness ranged from approx. 5 to 30 A for the carbide and from approx. 15 to 80 A for the carbon. The reflectivity measurements were made using Fe and Al K/sub α/ at grazing incidence. The emphasis in these studies is on the application of surface-analysis results in suggesting modifications to the fabrication process and in evaluating the results such modifications have on the layer stoichiometry, continuity, and periodicity of the dispersion elements so produced

  1. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    International Nuclear Information System (INIS)

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C222 1 and P4 1 2 1 2. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively. The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222 1 , with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P4 1 2 1 2, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme

  2. 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

  3. 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.

  4. 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°

  5. 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

  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. 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%

  8. 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.

  9. 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

  10. 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

  11. Crystal structure analysis of LaMnO_3 with x-ray diffraction technique using the Rietveld method

    International Nuclear Information System (INIS)

    Engkir Sukirman; Wisnu Ari Adi; Yustinus Purwamargapratala

    2010-01-01

    Crystal structure analysis of LaMnO_3 using the Rietveld method has been carried out. The LaMnO_3 sample was synthesized with high energy mechanical milling from the raw materials of La_2O_3 and MnO_2 with the appropriate mol ratio. Milling were performed for 10 hours, pelletized and hereinafter sintered at 1350 °C for 6 hours. The sample characterizations covered the crystal structure and electric-magnetic properties of the materials by X-ray diffraction technique using the Rietveld method and the four point probe, respectively. The Rietveld refinement results based on the X-rays diffraction data indicate that the sample of LaMnO_3 is single phase with the crystal system: orthorhombic, the space group: Pnma No. 62 and the lattice parameters: a = 55.4405(9) Å; b = 7.717(1) Å dan c = 5.537(1) Å. The material owns Magnetic Resonance (MR) respond of 7 %, the mean value of crystallite size, D = 17 nm and lattice strain, e = - 0.5 %. So, the material go through a compressive strain, and according to the Nanda's strain model, it becomes a type G antiferromagnetic insulator. Because the insulator properties of the material does not change although being hit by the external magnetic field, hence the MR respond is only caused by the order of electron spin. Therefore at room temperature, LaMnO_3_._0 just exhibits a small MR respond. (author)

  12. 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

  13. 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.

  14. Proton exciting X ray analysis

    International Nuclear Information System (INIS)

    Ma Xinpei

    1986-04-01

    The analyzing capability of proton exciting X ray analysis for different elements in organisms was discussed, and dealing with examples of trace element analysis in the human body and animal organisms, such as blood serum, urine, and hair. The sensitivity, accuracy, and capability of multielement analysis were discussed. Its strong points for the trace element analysis in biomedicine were explained

  15. 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

  16. 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°.

  17. Synthesis, characterization and single crystal X-ray analysis of chlorobis(N,N-dimethyldithiocarbamato-S,S′antimony(III

    Directory of Open Access Journals (Sweden)

    H.P.S. Chauhan

    2015-07-01

    Full Text Available The title compound chlorobis(N,N-dimethyldithiocarbamato-S,S′antimony(III has been prepared in distilled acetonitrile and characterized by physicochemical [melting point and molecular weight determination, elemental analysis (C, H, N, S & Sb], spectral [FT–IR, far IR, NMR (1H & 13C] studies. The crystal and molecular structure was further confirmed using single crystal X-ray diffraction analysis which features a five-coordinate geometry for antimony(III within a ClS4 donor set. The distortion in the co-planarity of ClSbS3 evidences the stereochemical influence exerts by the lone pair of electrons on antimony(III. Two centrosymmetrically related molecule held together via C–H···Cl secondary interaction result in molecular aggregation of the compound.

  18. 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

  19. 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

  20. 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

  1. 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%.

  2. Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

    International Nuclear Information System (INIS)

    Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

    2012-01-01

    The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

  3. 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

  4. 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

  5. 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

  6. X-ray system analysis

    International Nuclear Information System (INIS)

    Shapiro, J.S.

    1985-01-01

    An X-ray system tester for measuring anode voltage, cathode voltage, anode current, filament current and line voltage in an X-ray system has a selector which couples one of these analog signals or one of a plurality of processing control signals entered by an operator from a control panel to a digitizing section selectively in accordance with control signals provided to the selector by a computing section. The digitizing section converts the selected signal into a train of pulses having a frequency proportional to the value of the selected signal. These pulses are counted, the counts being used by the computing section to determine the value of the selected signal. This computed value is stored in a computing memory section of the computing section. The computing section is adapted to store a plurality of the sets of signals produced during a corresponding sequence of operational intervals of the X-ray system and determines a measure of the deviation of any selected one of the stored electrical signals over the sequence of operating intervals. Each signal produced during the sequential operational intervals can be recalled to aid analysis of the operation of the X-ray system. (author)

  7. 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°

  8. 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°

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Chemical state analysis of oxide thin films using a high resolution double crystal X-ray fluorescence spectrometer

    International Nuclear Information System (INIS)

    Masuda, Hirohisa; Morinaga, Kenji; Ohta, Yoshio.

    1995-01-01

    The chemical state analysis of r.f.-sputtered amorphous oxide thin films was determined by a high resolution X-ray fluorescence spectrometer with double crystals. The polymerization degree of silicate anions in the silicate film was as same as a target (α-Quartz). The oxygen coordination number of Al 3+ ions in the aluminate film was different from a target (α-Al 2 O 3 ), and it was a mixture of 4 and 6 in a spinel-like structure. In CaO-SiO 2 and CaO-Al 2 O 3 films, when the film thickness is thin at the beginning of sputtering, the composition of films are in the shortage of CaO. But when the film thickness become thicker, the composition of films become as same as the target. From the results above, the chemical state of films and their variations with film thickness can be clarified by using the apparatus. (author)

  15. High pressure single-crystal micro X-ray diffraction analysis with GSE_ADA/RSV software

    Science.gov (United States)

    Dera, Przemyslaw; Zhuravlev, Kirill; Prakapenka, Vitali; Rivers, Mark L.; Finkelstein, Gregory J.; Grubor-Urosevic, Ognjen; Tschauner, Oliver; Clark, Simon M.; Downs, Robert T.

    2013-08-01

    GSE_ADA/RSV is a free software package for custom analysis of single-crystal micro X-ray diffraction (SCμXRD) data, developed with particular emphasis on data from samples enclosed in diamond anvil cells and subject to high pressure conditions. The package has been in extensive use at the high pressure beamlines of Advanced Photon Source (APS), Argonne National Laboratory and Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. The software is optimized for processing of wide-rotation images and includes a variety of peak intensity corrections and peak filtering features, which are custom-designed to make processing of high pressure SCμXRD easier and more reliable.

  16. 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°.

  17. 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

  18. X-ray diffraction studies of NbTe 2 single crystal

    Indian Academy of Sciences (India)

    The composition of the grown crystals was confirmed on the basis of energy dispersive analysis by X-ray (EDAX) and remaining structural characterization was also accomplished by X-ray diffraction (XRD) studies. Lattice parameters, volume and X-ray density have been carried out for the grown crystals. The particle size ...

  19. Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

    Energy Technology Data Exchange (ETDEWEB)

    Hubin, Elizabeth A.; Lilic, Mirjana; Darst, Seth A.; Campbell, Elizabeth A.

    2017-07-13

    The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σA, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. X-ray Microprobe for Fluorescence and Diffraction Analysis

    International Nuclear Information System (INIS)

    Ice, G.E.

    2005-01-01

    X-ray diffraction (see unit 1.1) and x-ray excited fluorescence analysis are powerful techniques for the nondestructive measurement of crystal structure and chemical composition. X-ray fluorescence analysis is inherently nondestructive with orders of magnitude lower power deposited for the same detectable limit as with fluorescence excited by charged particle probes (Sparks, 1980). X-ray diffraction analysis is sensitive to crystal structure with orders-of-magnitude greater sensitivity to crystallographic strain than electron probes (Rebonato, et al. 1989). When a small-area x-ray microbeam is used as the probe, chemical composition (Z>14), crystal structure, crystalline texture, and crystalline strain distributions can be determined. These distributions can be studied both at the surface of the sample and deep within the sample (Fig. 1). Current state-of-the-art can achieve an ∼1 mm-D x-ray microprobe and an ∼0.1 mm-D x-ray microprobe has been demonstrated (Bilderback, et al., 1994). Despite their great chemical and crystallographic sensitivities, x-ray microprobe techniques have until recently been restricted by inefficient x-ray focusing optics and weak x-ray sources; x-ray microbeam analysis was largely superseded by electron techniques in the 50's. However, interest in x-ray microprobe techniques has now been revived (Howells, et al., 1983; Ice and Sparks, 1984; Chevallier, et al., 1997; Riekel 1992; Thompson, el al., 1992; and Making and Using... 1997) by the development of efficient x-ray focusing optics and ultra-high intensity synchrotron x-ray sources (Buras and Tazzari, 1984; Shenoy, et al., 1988). These advances have increased the achievable microbeam flux by more than 11 orders of magnitude (Fig. 2) (Ice, 1997); the flux in a tunable 1 mm-D beam on a 'so called' 3rd-generation synchrotron source such as the APS can exceed the flux in a fixed-energy mm2 beam on a conventional source. These advances make x-ray microfluorescence and x-ray

  5. 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

  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. 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

  12. 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°

  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. 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

  15. 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.

  16. LCP crystallization and X-ray diffraction analysis of VcmN, a MATE transporter from Vibrio cholerae

    Energy Technology Data Exchange (ETDEWEB)

    Kusakizako, Tsukasa [Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Tanaka, Yoshiki [Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192 (Japan); Hipolito, Christopher J. [Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575 (Japan); Kuroda, Teruo [Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Ishitani, Ryuichiro [Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Suga, Hiroaki [Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nureki, Osamu, E-mail: nureki@bs.s.u-tokyo.ac.jp [Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan)

    2016-06-22

    A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively. Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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).

  3. Crystallized solids characterization by X-ray diffraction

    International Nuclear Information System (INIS)

    Broll, N.

    1996-01-01

    This work deals with the crystallized solids characterization by X-ray diffraction. The powders diffraction principle is described. Then are given the different powders diffraction experimental methods. An X-ray diffraction device is essentially constituted of three parts: the X-rays source, the sample and the detector. The source is usually constituted by an X-rays tube whereas the sample can be fixed on a photographic chamber or put on a goniometer. The different photographic chambers which can be used (Debye-Scherrer, Seeman-Bohlin and Guinier) are described. The powders diffractometer the most used is a Bragg-Brentano focusing diffractometer because it allows to obtain very sharp spectral lines and an important diffracted intensity. The detectors which are the mainly used are the scintillation counters. The most important use in powders diffractometry is the identification of the different phases of a sample. The phases identification consists to compare the unknown sample spectrum at those of standard materials indexed until now. Two methods exist at present. They are explained and their limits in the phases search are given. Another use of the X-ray diffraction is the quantitative analysis. It consists to determine the concentrations of each crystal phases of a sample. The principles of these quantitative methods are given. The lattice parameters of a polycrystal material can be determined from its X-ray pattern too with a very high precision. The way to index powders patterns is given. The residual stresses of materials can also be estimated. The principle of this measured method is explained. It is at last possible to study from an X-ray pattern, the material grain orientations during the different steps of preparation and working. (O.M.). 13 refs., 19 figs., 1 tab

  4. 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

  5. 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.

  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 X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri

    International Nuclear Information System (INIS)

    Dobritzsch, Doreen; Andersen, Birgit; Piškur, Jure

    2005-01-01

    Dihydropyrimidinase from the yeast S. kluyveri was crystallized by vapour diffusion. The crystals belong to space group P2 1 (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 Å, β = 91.4°) and diffracted to 2.6 Å resolution. Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated β-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 Å at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2 1 (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 Å, β = 91.4°), with one homotetramer per asymmetric unit

  8. 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%

  9. 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.

  10. X-ray diffraction analysis of LiCu2O2 crystals with additives of silver atoms

    International Nuclear Information System (INIS)

    Sirotinkin, V. P.; Bush, A. A.; Kamentsev, K. E.; Dau, H. S.; Yakovlev, K. A.; Tishchenko, E. A.

    2015-01-01

    Silver-containing LiCu 2 O 2 crystals up to 4 × 8 × 8 mm in size were grown by the crystallization of 80(1-x)CuO · 20 x AgNO 3 · 20Li 2 CO 3 (0 ≤ x ≤ 0.5) mixture melt. According to the X-ray spectral and Rietveld X-ray diffraction data, the maximum amount of silver incorporated in the LiCu 2 O 2 structure is about 4 at % relative to the copper content. It was established that silver atoms occupy statistically crystallographic positions of lithium atoms. The incorporation of silver atoms is accompanied by a noticeable increase in parameter c of the LiCu 2 O 2 rhombic unit cell, a slight increase in parameter a, and a slight decrease in parameter b

  11. 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

  12. 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.

  13. 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)

  14. 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

  15. 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

  16. Full analysis of feldspar texture and crystal structure by combining X-ray and electron techniques

    DEFF Research Database (Denmark)

    Balic Zunic, Tonci; Piazolo, Sandra; Katerinopoulou, Anna

    2013-01-01

    identification plus crystal structure refinement of all components in a feldspar intergrowth. This procedure was applied to perthitic intergrowths in feldspars from two different pegmatites in the Larvik plutonic complex in the southern part of the Oslo region, Norway. It revealed that the two starting high...

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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%

  8. 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

  9. 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.

  10. 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

  11. 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%

  12. 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

  13. 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.

  14. 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°

  15. 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

  16. 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

  17. 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)

  18. 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

  19. 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

  20. 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.

  1. 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%.

  2. 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%

  3. 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°

  4. 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

  5. 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°

  6. 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

  7. Crystallization and X-ray diffraction analysis of human CLEC5A (MDL-1), a dengue virus receptor

    International Nuclear Information System (INIS)

    Watson, Aleksandra A.; O’Callaghan, Christopher A.

    2009-01-01

    Recombinant human CLEC5A was crystallized in the trigonal space group P3 1 and X-ray diffraction data were collected to 1.56 Å resolution. The human C-type lectin-like protein CLEC5A (also known as MDL-1) is expressed on the surface of myeloid cells and plays a critical role in dengue-virus-induced disease by signalling through the transmembrane adaptor protein DAP12. The C-type lectin-like domain of CLEC5A was expressed in Escherichia coli, refolded and purified. Recombinant CLEC5A crystals were grown by sitting-drop vapour diffusion using polyethylene glycol 6000 as a precipitant. After optimization, crystals were grown which diffracted to 1.56 Å using synchrotron radiation. The results presented in this paper suggest that crystals producing diffraction of this quality will be suitable for structural determination of human CLEC5A

  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. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of glucuronic acid dehydrogenase from Chromohalobacter salexigens

    International Nuclear Information System (INIS)

    Ahn, Jae-Woo; Lee, Shin Youp; Kim, Sangwoo; Cho, Sun Ja; Lee, Sun Bok; Kim, Kyung-Jin

    2011-01-01

    Recombinant glucuronic acid dehydrogenase from the halophilic bacterium Chromohalobacter salexigens has been crystallized and X-ray diffraction data collected to a maximum resolution of 2.1 Å. Glucuronic acid dehydrogenase (GluUADH), the product of the Csal-2474 gene from the halophilic bacterium Chromohalobacter salexigens DSM 3043, is an enzyme with potential use in the conversion of glucuronic acid in seaweed biomass to fuels and chemicals. GluUADH is an enzyme that catalyzes the oxidation of glucuronic acid (GluUA) and galacturonic acid (GalUA) and has a preference for NAD + rather than NADP + as a cofactor. Recombinant GluUADH was crystallized in the presence of 0.2 M calcium acetate, 0.1 M Tris–HCl pH 7.0 and 20% PEG 3000 at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The GluUADH crystal belonged to space group P6 3 , with unit-cell parameters a = b = 122.58, c = 150.49 Å, γ = 120°. With one molecule per asymmetric unit, the crystal volume per unit protein weight (V M ) is 2.78 Å 3 Da −1 . The structure was solved by the single anomalous dispersion method and structure refinement is in progress

  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. 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

  13. 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.

  14. 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

  15. 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.

  16. 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°

  17. 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

  18. 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

  19. 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.

  20. 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°

  1. 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°

  2. Radionuclide X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Cechak, T.

    1994-01-01

    The author's achievements in the title field are summarized and discussed. The following topics are dealt with: (i) principles of radionuclide X-ray fluorescence analysis; (ii) mathematical methods in X-ray fluorescence analysis; (iii) Ross differential filters; (iv) application of radionuclide X-ray fluorescence analysis in the coal industry (with emphasis on the determination of the ash content, sulfur content, and arsenic content of coal); and (v) evaluation of the X-ray fluorescence analyzer from the radiological safety point of view. (P.A.)

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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%

  13. 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%.

  14. 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

  15. 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

  16. 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.

  17. Proton induced X-ray emission analysis

    International Nuclear Information System (INIS)

    Khan, Rashiduzzman

    1976-09-01

    The developments in proton induced X-ray emission analysis are reviewed. Techniques for analyzing thick and thin samples of different origin are described. Discussions on the application of proton induced X-ray emission analysis in different fields, comparison of the sensitivity of this method with other analytical techniques, its limitations and possible improvements are presented

  18. 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.

  19. 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

  20. 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)

  1. 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

  2. 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°

  3. 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

  4. 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°

  5. 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°.

  6. 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)

  7. 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

  8. 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%.

  9. 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

  10. 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 )

  11. Automated x-ray fluorescence analysis

    International Nuclear Information System (INIS)

    O'Connell, A.M.

    1977-01-01

    A fully automated x-ray fluorescence analytical system is described. The hardware is based on a Philips PW1220 sequential x-ray spectrometer. Software for on-line analysis of a wide range of sample types has been developed for the Hewlett-Packard 9810A programmable calculator. Routines to test the system hardware are also described. (Author)

  12. Image analysis in x-ray cinefluorography

    Energy Technology Data Exchange (ETDEWEB)

    Ikuse, J; Yasuhara, H; Sugimoto, H [Toshiba Corp., Kawasaki, Kanagawa (Japan)

    1979-02-01

    For the cinefluorographic image in the cardiovascular diagnostic system, the image quality is evaluated by means of MTF (Modulation Transfer Function), and object contrast by introducing the concept of x-ray spectrum analysis. On the basis of these results, further investigation is made of optimum X-ray exposure factors set for cinefluorography and the cardiovascular diagnostic system.

  13. Crystallization and X-ray data analysis of the 10 kDa C-terminal lid subdomain from Caenorhabditis elegans Hsp70

    Energy Technology Data Exchange (ETDEWEB)

    Worrall, Liam; Walkinshaw, Malcolm D., E-mail: m.walkinshaw@ed.ac.uk [School of Biological Sciences, University of Edinburgh, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JR,Scotland (United Kingdom)

    2006-09-01

    Crystals of the C-terminal 10 kDa lid subdomain from the C. elegans chaperone Hsp70 have been obtained that diffract X-rays to ∼3.5 Å and belong to space group I2{sub 1}2{sub 1}2{sub 1}. Analysis of X-ray data and initial heavy-atom phasing reveals 24 monomers in the asymmetric unit related by 432 non-crystallographic symmetry. Hsp70 is an important molecular chaperone involved in the regulation of protein folding. Crystals of the C-terminal 10 kDa helical lid domain (residues 542–640) from a Caenorhabditis elegans Hsp70 homologue have been produced that diffract X-rays to ∼3.4 Å. Crystals belong to space group I2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 197, c = 200 Å. The Matthews coefficient, self-rotation function and Patterson map indicate 24 monomers in the asymmetric unit, showing non-crystallographic 432 symmetry. Molecular-replacement studies using the corresponding domain from rat, the only eukaryotic homologue with a known structure, failed and a mercury derivative was obtained. Preliminary MAD phasing using SHELXD and SHARP for location and refinement of the heavy-atom substructure and SOLOMON for density modification produced interpretable maps with a clear protein–solvent boundary. Further density-modification, model-building and refinement are currently under way.

  14. 2-nitrobenzohydrazide as a potent urease inhibitor: synthesis, characterization and single crystal x-ray diffraction analysis

    International Nuclear Information System (INIS)

    Abbas, N.; Khan, I.; Hameed, S.; Batool, S.

    2018-01-01

    2-Nitrobenzohydrazide was efficiently synthesized in two steps by the esterification of 2-nitrobenzoic acid followed by the treatment with hydrazine hydrate in methanol. The structure of 2-nitrobenzohydrazide was established by modern spectro-analytical techniques including FTIR, 1H and 13C-NMR spectroscopy and unequivocally confirmed by single crystal X-ray diffraction data. 2-Nitrobenzohydrazide crystallized in orthorhombic space group P 21 21 21 with unit cell dimensions a = 4.9764(4) Å, b = 12.5280 (3) Å, c = 12.8512(1) Å, α = β = γ = 90°. A combination of N‒H…N and N‒H…O hydrogen bonds stabilized the crystal packing of 3. The synthesized compound 3 was assessed as urease inhibitor against Jack bean urease and the results revealed good inhibitory potency with an IC50 value of 4.25 ± 0.08 µM. This inhibition strength was 5-fold higher compared to the reference inhibitor thiourea (IC50 = 21.00 ± 0.11 µM). The molecular docking studies of the synthesized inhibitor 3 inside the active pocket of urease revealed several important binding interactions. (author)

  15. 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

  16. 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

  17. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of D-lactate dehydrogenase from Lactobacillus jensenii.

    Science.gov (United States)

    Kim, Sangwoo; Kim, Yong Hwan; Kim, Kyung-Jin

    2014-08-01

    The thermostable D-lactate dehydrogenase from Lactobacillus jensenii (LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD(+). The polymers of lactic acid are used as biodegradable bioplastics. The LjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mM Tris-HCl pH 9, 200 mM magnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The crystal belonged to space group P3121, with unit-cell parameters a = b = 90.5, c = 157.8 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 Å(3) Da(-1), which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative.

  18. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

    Science.gov (United States)

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-03-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

  19. 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.

  20. Preparation and Crystal Structures of Some AIVB2IIO4 Compounds: Powder X-Ray Diffraction and Rietveld Analysis

    Directory of Open Access Journals (Sweden)

    K. Jeyadheepan

    2014-01-01

    Full Text Available The AIVB2IIO4 compounds such as cadmium tin oxide (Cd2SnO4 or CTO and zinc tin oxide (Zn2SnO4 or ZTO are synthesized by solid state reaction of the subsequent binary oxides. The synthesized powders were analyzed through the powder X-ray diffraction (PXRD. Cell search done on the PXRD patterns shows that the Cd2SnO4 crystallizes in orthorhombic structure with space group Pbam and the cell parameters as a=5.568(2 Å, b=9.894(3 Å, and c=3.193(1 Å and the Zn2SnO4 crystallizes as cubic with the space group Fd3 -m and with the cell parameter a=8.660(2 Å. Rietveld refinement was done on the PXRD patterns to get the crystal structure of the Cd2SnO4 and Zn2SnO4 and to define the site deficiency of atoms which causes the electrical properties of the materials.

  1. Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design

    International Nuclear Information System (INIS)

    MacElrevey, Celeste; Wedekind, Joseph E.

    2005-01-01

    Well diffracting decamer crystals of the hepatitis delta virus RNA-editing site were prepared, but exhibited merohedral twinning and base averaging owing to duplex symmetry. A longer asymmetric construct that includes additional flanking RNA sequences has been crystallized that does not appear to exhibit these defects. RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ‘structure-based’ readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 Å resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 × 1 internal loops yielded crystals in the primitive tetragonal space group P4 1 2 1 2 or P4 3 2 1 2. X-ray diffraction data were collected to 2.8 Å resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 Å. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported

  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. Synchrotron x-ray microbeam characteristics for x-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Iida, Atsuo; Noma, Takashi

    1995-01-01

    X-ray fluorescence analysis using a synchrotron x-ray microprobe has become an indispensable technique for non-destructive micro-analysis. One of the most important parameters that characterize the x-ray microbeam system for x-ray fluorescence analysis is the beam size. For practical analysis, however, the photon flux, the energy resolution and the available energy range are also crucial. Three types of x-ray microbeam systems, including monochromatic and continuum excitation systems, were compared with reference to the sensitivity, the minimum detection limit and the applicability to various types of x-ray spectroscopic analysis. 16 refs., 5 figs

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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%

  13. X-ray dosimetry of TlGaSe2 single crystals

    International Nuclear Information System (INIS)

    Kerimova, E.M.; Mustafaeva, S.N.; Mamedbeili, S.D.; Jabarov, J.N.; Iskenderova, P.M.; Kazimov, S.B.

    2002-01-01

    TlGaSe 2 compound belongs to group of layered semiconductors of A 3 B 3 C 2 6 -type. Photoelectric and optical properties of TlGaSe 2 single crystals were investigated in detail. Influence of gamma-, electron and neutron radiation on photoelectric properties of TlGaSe 2 single crystals is investigated too. The present work deals with experimental results relative to X-ray dosimetric characteristics of TlGaSe 2 crystals at 300 K. X-ray conductivity and X-ray dosimetric characteristic measurements are carried out in low load resistance regime. The source of X-ray radiation is the installation of X-ray diffraction analysis (URS-55a) with the BCV-2(Cu). Intensity of X-ray radiation (E) is regulated by measurement with current variation in tube at each given value of X-ray radiation dose E (R/min) are measured by crystal dosimeter DRGZ-02. X-ray conductivity coefficients K σ characterising X-ray sensitivity of investigated crystals are determined as the relative change of conductivity under X-ray radiation a per dose. There have been determined values of characteristic coefficients of TlGaSe 2 single crystal X-ray conductivity at different values of accelerating voltage (V a ) on the tube and corresponding doses of X-ray radiation. Analysis of obtained data showed that X-ray conductivity coefficients K σ in studied crystals are regularly decreased (from 0.276 to 0.033) as with the rise of dose (E=0.75-78.0 R/min) as with the increase of values of V a on X-ray tube (V a =254-50 keV). One of the possible reasons of observed regularities is that X-ray conductivity in investigated crystals, especially at comparatively low V a is due predominantly to radiation of thin layer of crystal. In this case with the rise of radiation intensity there have been started to prevail the mechanism of surface quadratic recombination which leads to observed decrease of X-ray conductivity. With the rise of accelerating potential 'effective hardness' is increased, as a result of which there

  14. Energy-dispersive x-Ray Analysis of Phosphorus, Potassium, Magnesium, and Calcium in Globoid Crystals in Protein Bodies from Different Regions of Cucurbita maxima Embryos 1

    Science.gov (United States)

    Lott, John N. A.; Greenwood, John S.; Vollmer, Catherine M.; Buttrose, Mark S.

    1978-01-01

    The seeds of Cucurbita maxima contain protein bodies with electrondense globoid crystals. Because of their density globoid crystals are ideal material for energy-dispersive x-ray (EDX) analysis studies of elemental composition. Fixation trials were carried out to test globoid crystal extraction during glutaraldehyde fixation, water washing, and ethanol dehydration. Glutaraldehyde fixation without subsequent washing or dehydration alone produced no significant changes in elemental composition of cotyledon globoid crystals. If glutaraldehyde fixation was followed by water washes or ethanol dehydration there was some loss of the major globoid crystal elements but the relative percentages of the elements P, K, Ca, and Mg remained relatively unchanged. In this paper results of a study of the P, K, Mg, and Ca content of globoid crystals in different tissues of squash embryos are presented. The globoid crystals in the radicle were found to be the least dense in the embryo. Globoid crystals from all embryo regions contained P, K, and Mg. In the various embryo regions P and Mg maintained relatively constant proportions of the globoid crystal composition while K and Ca varied. Of particular significance is the distribution of Ca which is generally an immobile element. Calcium was found in highest amounts in the globoid crystals of the radicle and stem regions while globoid crystals in much of the cotyledon contained little, if any, Ca. The Ca storage thus seems to be spatially arranged in a manner that would aid early growth of the root-shoot axis. PMID:16660439

  15. 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.

  16. 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

  17. 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

  18. Synthesis, characterization and single crystal x-ray analysis of a complex of iron(II) bis(2,4-dimethylphenyl)dithiophosphate with 4-ethylpyridine

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Sandeep; Andotra, Savit; Kaur, Mandeep [University of Jammu, Department of Chemistry (India); Gupta, Vivek K.; Kant, Rajni [Department of Physics and Electronics, University of Jammu, X-ray Crystallographic Laboratory (India); Pandey, Sushil K., E-mail: kpsushil@rediffmail.com [University of Jammu, Department of Chemistry (India)

    2016-09-15

    Complex of iron(II) bis(2,4-dimethylphenyl)dithiophosphate with 4-ethylpyridine [((2,4- (CH{sub 3}){sub 2}C{sub 6}H{sub 3}O)2PS2)2Fe(NC{sub 5}H{sub 4}(C{sub 2}H{sub 5})-4){sub 2}] is synthesized and characterized by elemental analysis, magnetic moment, IR spectroscopy and single crystal X-ray analysis. Complex crystallizes in the monoclinic sp. gr. P2{sub 1}/n, Z = 2. Crystal structure consists of mononuclear units with Fe(II) ion chelated by four S atoms of the two diphenyldithiophosphate ligands in bidentate manner. N atoms from two 4-ethylpyridine ligands are axially coordinated to the Fe(II) atom leading to an octahedral geometry.

  19. 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).

  20. VETA-I x ray test analysis

    Science.gov (United States)

    Brissenden, R. J. V.; Chartas, G.; Freeman, M. D.; Hughes, J. P.; Kellogg, E. M.; Podgorski, W. A.; Schwartz, D. A.; Zhao, P.

    1992-01-01

    This interim report presents some definitive results from our analysis of the VETA-I x-ray testing data. It also provides a description of the hardware and software used in the conduct of the VETA-I x-ray test program performed at the MSFC x-ray Calibration Facility (XRCF). These test results also serve to supply data and information to include in the TRW final report required by DPD 692, DR XC04. To provide an authoritative compendium of results, we have taken nine papers as published in the SPIE Symposium, 'Grazing Incidence X-ray/EUV Optics for Astronomy and Projection Lithography' and have reproduced them as the content of this report.

  1. 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.

  2. 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%.

  3. 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%

  4. 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.

  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. 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

  7. X-ray scattering from surfaces of organic crystals

    DEFF Research Database (Denmark)

    Gidalevitz, D.; Feidenhans'l, R.; Smilgies, D.-M.

    1997-01-01

    X-ray scattering experiments have been performed on the surfaces of organic crystals. The (010) cleavage planes of beta-alanine and alpha-glycine were investigated, and both specular and off-specular crystal truncation rods were measured. This allowed a determination of the molecular layering...

  8. 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

  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. A short working distance multiple crystal x-ray spectrometer

    Science.gov (United States)

    Dickinson, B.; Seidler, G.T.; Webb, Z.W.; Bradley, J.A.; Nagle, K.P.; Heald, S.M.; Gordon, R.A.; Chou, I.-Ming

    2008-01-01

    For x-ray spot sizes of a few tens of microns or smaller, a millimeter-sized flat analyzer crystal placed ???1 cm from the sample will exhibit high energy resolution while subtending a collection solid angle comparable to that of a typical spherically bent crystal analyzer (SBCA) at much larger working distances. Based on this observation and a nonfocusing geometry for the analyzer optic, we have constructed and tested a short working distance (SWD) multicrystal x-ray spectrometer. This prototype instrument has a maximum effective collection solid angle of 0.14 sr, comparable to that of 17 SBCA at 1 m working distance. We find good agreement with prior work for measurements of the Mn K?? x-ray emission and resonant inelastic x-ray scattering for MnO, and also for measurements of the x-ray absorption near-edge structure for Dy metal using L??2 partial-fluorescence yield detection. We discuss future applications at third- and fourth-generation light sources. For concentrated samples, the extremely large collection angle of SWD spectrometers will permit collection of high-resolution x-ray emission spectra with a single pulse of the Linac Coherent Light Source. The range of applications of SWD spectrometers and traditional multi-SBCA instruments has some overlap, but also is significantly complementary. ?? 2008 American Institute of Physics.

  11. Crystallization and X-ray diffraction analysis of an ‘all-locked’ nucleic acid duplex derived from a tRNASer microhelix

    International Nuclear Information System (INIS)

    Behling, Katja; Eichert, André; Fürste, Jens P.; Betzel, Christian; Erdmann, Volker A.; Förster, Charlotte

    2009-01-01

    A completely ‘all-locked’ nucleic acid duplex was designed from an E. coli tRNA Ser microhelix. The helix consists exclusively of LNA building blocks and was crystallized. The crystals diffracted to 1.9 Å resolution. Modified nucleic acids are of great interest with respect to their nuclease resistance and enhanced thermostability. In therapeutical and diagnostic applications, such molecules can substitute for labile natural nucleic acids that are targeted against particular diseases or applied in gene therapy. The so-called ‘locked nucleic acids’ contain modified sugar moieties such as 2′-O,4′-C-methylene-bridged β-d-ribofuranose and are known to be very stable nucleic acid derivatives. The structure of locked nucleic acids in single or multiple LNA-substituted natural nucleic acids and in LNA–DNA or LNA–RNA heteroduplexes has been well investigated, but the X-ray structure of an ‘all-locked’ nucleic acid double helix has not been described to date. Here, the crystallization and X-ray diffraction data analysis of an ‘all-locked’ nucleic acid helix, which was designed as an LNA originating from a tRNA Ser microhelix RNA structure, is presented. The crystals belonged to space group C2, with unit-cell parameters a = 77.91, b = 40.74, c = 30.06 Å, β = 91.02°. A high-resolution and a low-resolution data set were recorded, with the high-resolution data showing diffraction to 1.9 Å resolution. The crystals contained two double helices per asymmetric unit, with a Matthews coefficient of 2.48 Å 3 Da −1 and a solvent content of 66.49% for the merged data

  12. Purification, crystallization and X-ray diffraction analysis of a novel ring-cleaving enzyme (BoxCC) from Burkholderia xenovorans LB400

    International Nuclear Information System (INIS)

    Bains, Jasleen; Boulanger, Martin J.

    2008-01-01

    Preliminary X-ray diffraction studies of a novel ring-cleaving enzyme from B. xenovorans LB400 encoded by the benzoate-oxidation (box) pathway. The assimilation of aromatic compounds by microbial species requires specialized enzymes to cleave the thermodynamically stable ring. In the recently discovered benzoate-oxidation (box) pathway in Burkholderia xenovorans LB400, this is accomplished by a novel dihydrodiol lyase (BoxC C ). Sequence analysis suggests that BoxC C is part of the crotonase superfamily but includes an additional uncharacterized region of approximately 115 residues that is predicted to mediate ring cleavage. Processing of X-ray diffraction data to 1.5 Å resolution revealed that BoxC C crystallized with two molecules in the asymmetric unit of the P2 1 2 1 2 1 space group, with a solvent content of 47% and a Matthews coefficient of 2.32 Å 3 Da −1 . Selenomethionine BoxC C has been purified and crystals are currently being refined for anomalous dispersion studies

  13. A 1:1 pharmaceutical cocrystal of myricetin in combination with uncommon piracetam conformer: X-ray single crystal analysis and mechanochemical synthesis

    Science.gov (United States)

    Sowa, Michał; Ślepokura, Katarzyna; Matczak-Jon, Ewa

    2014-01-01

    Combination of two Active Pharmaceutical Ingredients, myricetin and piracetam, yields a 1:1 cocrystal characterized by X-ray single-crystal and powder diffraction, Raman spectroscopy, 1H NMR, thermal analysis (DSC and TG-DTA) methods. Constituents of the cocrystalline phase were also investigated in terms of Hirshfeld surfaces. Compounds in their neutral forms cocrystallize in the Pna21 space group of orthorhombic system. Notably, piracetam adopts an uncommon conformation, not encountered in its cocrystals previously described. In the crystal lattice, a three-dimensional hydrogen-bonded network is observed, including formation of a 2D molecular scaffolding motif. A scale-up procedure is readily available with use of solvent-drop grinding method, in which application of a variety of common solvents leads to formation of the cocrystal, as confirmed by XRPD and Raman spectroscopy.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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%

  20. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    Science.gov (United States)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  1. Capacity of X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Wobrauschek, P.; Kregsamer, P.

    1997-01-01

    X-Ray fluorescence analysis (XRF) is a powerful analytical tool for the qualitative and quantitative determination of chemical elements in a sample. Two different detection principles are accepted widely: wavelength dispersive and energy dispersive. Various sources for XRF are discussed: X-ray tubes, accelerators for particle induced XRF, radioactive isotopes, and the use of synchrotron radiation. Applications include environmental, technical, medical, fine art, and forensic studies. Due to the demands of research and application special techniques like total reflection XRF (TXRF) were developed with ultimately achievable detection limits in the femtogram region. The elements detectable by XRF range from Be to U. (author)

  2. 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

  3. Kinematic X-Ray Analysis Apparatus

    NARCIS (Netherlands)

    Koningsberger, D.C.; Brinkgreve, P.

    1983-01-01

    In an X-ray analysis apparatus, a moving mechanism is provided by a main guide member along which a main slide device can be displaced. Rotatably connected with the main slide device is a detector guide member along which a detection slide device is displaced. The main slide device, as well as the

  4. Quantitative X-ray analysis of pigments

    International Nuclear Information System (INIS)

    Araujo, M. Marrocos de

    1987-01-01

    The 'matrix-flushing' and the 'adiabatic principle' methods have been applied for the quantitative analysis through X-ray diffraction patterns of pigments and extenders mixtures, frequently used in paint industry. The results obtained have shown the usefulness of these methods, but still ask for improving their accuracy. (Author) [pt

  5. 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

  6. 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%

  7. 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%.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 Å resolution using a twinned crystal

    International Nuclear Information System (INIS)

    Mizutani, Kimihiko; Toyoda, Mayuko; Sagara, Kenta; Takahashi, Nobuyuki; Sato, Atsuko; Kamitaka, Yuji; Tsujimura, Seiya; Nakanishi, Yuji; Sugiura, Toshiyuki; Yamaguchi, Shotaro; Kano, Kenji; Mikami, Bunzo

    2010-01-01

    The crystal structure of bilirubin oxidase (BOD) from M. verrucaria has been determined at 2.3 Å resolution using a merohedrally twinned crystal. BOD has four copper-coordination sites that are almost identical to those of other multicopper oxidases and is also very similar to them in overall structure. Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6 3 . Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33–2.3 Å. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD

  19. 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

  20. Preparation of specimens for analysis by: X-ray diffraction and X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Banos L, L.

    2004-01-01

    Specimen preparation is one of the most important requirements in the analysis of samples by X-ray Diffraction and X-ray Fluorescence. This statement is especially true for samples containing different types of materials. There are many forms of specimen suitable for X-ray analysis and the type of the sample as received will generally determine the method of pretreatment. It is convenient to refer to the material received for analysis as the sample, and that, which is actually analyzed as the specimen. The powder Diffraction method assumes that the particles in the specimen are ideally random orientation and that there are enough crystallites in the specimen to achieve a representative intensity distribution for these crystallites. X ray Fluorescence is essentially a comparative method of analysis, it is vital that all standards and unknowns be presented to the spectrometer in a reproducible and identical manner. (Author) 3 refs., 6 figs

  1. Scattering of x-ray from crystal surfaces

    International Nuclear Information System (INIS)

    Andrews, S.R.; Cowley, R.A.

    1985-01-01

    X-ray measurements performed on a variety of materials demonstrate that it is possible to observe diffuse scattering that originates in the abrupt change of density at a crystal surface. Such a discontinuity gives rise, in general, to rods of scattering in reciprocal space which are most intense close to the Bragg peaks tau and are well defined for sufficiently smooth surfaces. For wave-vector transfer Q=tau+q the q-dependence of the intensity of scattering gives information on the topographic structure of the crystal surface. Experimental results on crystals of GaAs and KTaO 3 , with surfaces prepared in various ways, were obtained using conventional x-ray techniques with a rotating anode source and can be described by a continuum model of the surface. There are discrepancies between the predictions of the models and the experimental results and the suggest that further experiments are needed to achieve a more complete understanding. (author)

  2. A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

    Energy Technology Data Exchange (ETDEWEB)

    Trindade, Inês B.; Fonseca, Bruno M. [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal); Matias, Pedro M. [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal); Instituto de Biologia Experimental e Tecnológica (iBET), Apartado 12, 2780-901 Oeiras (Portugal); Louro, Ricardo O.; Moe, Elin, E-mail: elinmoe@itqb.unl.pt [Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras (Portugal)

    2016-08-09

    The gene encoding a putative siderophore-interacting protein from the marine bacterium S. frigidimarina was successfully cloned, followed by expression and purification of the gene product. Optimized crystals diffracted to 1.35 Å resolution and preliminary crystallographic analysis is promising with respect to structure determination and increased insight into the poorly understood molecular mechanisms underlying iron acquisition. Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacterium Shewanella frigidimarina NCIMB 400, the gene tagged as SFRI-RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 48.04, b = 78.31, c = 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.

  3. Total reflection X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Michaelis, W.; Prange, A.

    1987-01-01

    In the past few years, total reflection X-ray flourescence analysis (TXRF) has found an increasing number of assignments and applications. Experience of trace element analysis using TXRF and examples of applications are already widespread. Therefore, users of TXRF had the opportunity of an intensive exchange of their experience at the 1st workshop on total reflection X-ray fluorescence analysis which took place on May 27th and 28th 1986 at the GKSS Research Centre at Geesthacht. In a series of lectures and discussions dealing with the analytical principle itself, sample preparation techniques and applications as well as comuter programs for spectrum evaluation, the present state of development and the range of applications were outlined. 3 studies out of a total of 14 were included separately in the INIS and ENERGY databases. With 61 figs., 12 tabs [de

  4. 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

  5. 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

  6. 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

  7. 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)

  8. 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%

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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...

  17. 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

  18. 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

  19. 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

  20. Crystal defect studies using x-ray diffuse scattering

    International Nuclear Information System (INIS)

    Larson, B.C.

    1980-01-01

    Microscopic lattice defects such as point (single atom) defects, dislocation loops, and solute precipitates are characterized by local electronic density changes at the defect sites and by distortions of the lattice structure surrounding the defects. The effect of these interruptions of the crystal lattice on the scattering of x-rays is considered in this paper, and examples are presented of the use of the diffuse scattering to study the defects. X-ray studies of self-interstitials in electron irradiated aluminum and copper are discussed in terms of the identification of the interstitial configuration. Methods for detecting the onset of point defect aggregation into dislocation loops are considered and new techniques for the determination of separate size distributions for vacancy loops and interstitial loops are presented. Direct comparisons of dislocation loop measurements by x-rays with existing electron microscopy studies of dislocation loops indicate agreement for larger size loops, but x-ray measurements report higher concentrations in the smaller loop range. Methods for distinguishing between loops and three-dimensional precipitates are discussed and possibilities for detailed studies considered. A comparison of dislocation loop size distributions obtained from integral diffuse scattering measurements with those from TEM show a discrepancy in the smaller sizes similar to that described above

  1. Crystal defect studies using x-ray diffuse scattering

    Energy Technology Data Exchange (ETDEWEB)

    Larson, B.C.

    1980-01-01

    Microscopic lattice defects such as point (single atom) defects, dislocation loops, and solute precipitates are characterized by local electronic density changes at the defect sites and by distortions of the lattice structure surrounding the defects. The effect of these interruptions of the crystal lattice on the scattering of x-rays is considered in this paper, and examples are presented of the use of the diffuse scattering to study the defects. X-ray studies of self-interstitials in electron irradiated aluminum and copper are discussed in terms of the identification of the interstitial configuration. Methods for detecting the onset of point defect aggregation into dislocation loops are considered and new techniques for the determination of separate size distributions for vacancy loops and interstitial loops are presented. Direct comparisons of dislocation loop measurements by x-rays with existing electron microscopy studies of dislocation loops indicate agreement for larger size loops, but x-ray measurements report higher concentrations in the smaller loop range. Methods for distinguishing between loops and three-dimensional precipitates are discussed and possibilities for detailed studies considered. A comparison of dislocation loop size distributions obtained from integral diffuse scattering measurements with those from TEM show a discrepancy in the smaller sizes similar to that described above.

  2. Preliminary X-ray analysis of twinned crystals of the Q88Y25-Lacpl esterase from Lactobacillus plantarum WCFS1

    International Nuclear Information System (INIS)

    Álvarez, Yanaisis; Esteban-Torres, María; Acebrón, Iván; Rivas, Blanca de las; Muñoz, Rosario; Martínez-Ripoll, Martín; Mancheño, José M.

    2011-01-01

    The Q88Y25-Lacpl esterase from L. plantarum WCFS1 has been recombinantly expressed, purified and crystallized. A native diffraction data set has been collected to 2.24 Å resolution. Q88Y25-Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His 6 -tagged Q88Y25-Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His 6 -tagged Q88Y25-Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å

  3. 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

  4. Variable-metric diffraction crystals for x-ray optics

    International Nuclear Information System (INIS)

    Smither, R.K.; Fernandez, P.B.

    1992-01-01

    A variable-metric (VM) crystal is one in which the spacing between the crystalline planes changes with position in the crystal. This variation can be either parallel to the crystalline planes or perpendicular to the crystalline planes of interest and can be produced by either introducing a thermal gradient in the crystal or by growing a crystal made of two or more elements and changing the relative percentages of the two elements as the crystal is grown. A series of experiments were performed in the laboratory to demonstrate the principle of the variable-metric crystal and its potential use in synchrotron beam lines. One of the most useful applications of the VM crystal is to increase the number of photons per unit bandwidth in a diffracted beam without losing any of the overall intensity. In a normal synchrotron beam line that uses a two-crystal monochromator, the bandwidth of the diffracted photon beam is determined by the vertical opening angle of the beam which is typically 0.10--0.30 mrad or 20--60 arcsec. When the VM crystal approach is applied, the bandwidth of the beam can be made as narrow as the rocking curve of the diffracting crystal, which is typically 0.005--0.050 mrad or 1--10 arcsec. Thus a very large increase of photons per unit bandwidth (or per unit energy) can be achieved through the use of VM crystals. When the VM principle is used with bent crystals, new kinds of x-ray optical elements can be generated that can focus and defocus x-ray beams much like simple lenses where the focal length of the lens can be changed to match its application. Thus both large magnifications and large demagnifications can be achieved as well as parallel beams with narrow bandwidths

  5. Molecular orbital study of the primary electron donor P700 of photosystem I based on a recent X-ray single crystal structure analysis

    International Nuclear Information System (INIS)

    Plato, Martin; Krauss, Norbert; Fromme, Petra; Lubitz, Wolfgang

    2003-01-01

    The X-ray structure analysis of photosystem (PS) I single crystals showed that the primary electron donor P700 is a heterodimer formed by one chlorophyll (Chl) a and one Chl a ' [Nature 411 (2001) 909]. The electronic structure of the cation radical P700 +· of the primary donor, which is created in the charge separation process, has been probed by semiempirical molecular orbital calculations including spin polarization effects (RHF-INDO/SP). The calculations, which were based on the X-ray structure, clearly show that P700 is a supermolecule formed by two chlorophyll species. They furthermore predict an asymmetrical charge and spin density distribution in favor of the monomeric Chl a half of this dimer in accordance with results from earlier EPR and ENDOR studies [J. Phys. Chem. B 105 (2000) 1225]. The stepwise inclusion of various electrostatic interactions of the dimer with its nearest surrounding (one threonine forming a hydrogen bond to the keto group of Chl a ' and two histidines liganding the Mg atoms of the two chlorophylls) leads to a systematic enhancement of this electronic asymmetry yielding a spin density ratio of almost 5:1 as also found experimentally. A large part of this value is caused by spin polarization effects. This result is only weakly affected by the electrostatic field of more remote amino acid residues and other pigment molecules ('accessory' Chl a molecules) present in PS I. A separate group of calculations involving local geometry optimizations by energy minimization techniques yields a further enhancement of the spin density asymmetry. A particularly strong effect is obtained by allowing for variations of the geometry of the vinyl groups on both chlorophylls of the P700 dimer. Theoretical results for individual isotropic proton and nitrogen hyperfine coupling constants, showing a satisfactory agreement with experimental findings, are also presented

  6. 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%

  7. 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

  8. 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

  9. 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

  10. 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

  11. X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

    DEFF Research Database (Denmark)

    Yennawar, Hemant; Møller, Magda; Gillilan, Richard

    2011-01-01

    The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer...

  12. 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

  13. 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)

  14. 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

  15. 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

  16. New structural studies of liquid crystal by reflectivity and resonant X-ray diffraction

    International Nuclear Information System (INIS)

    Fernandes, P.

    2007-04-01

    This memory presents three structural studies of smectic Liquid Crystals by reflectivity and resonant diffraction of X-rays. It is divided in five chapters. In the first a short introduction to Liquid Crystals is given. In particular, the smectic phases that are the object of this study are presented. The second chapter is consecrated to the X-ray experimental techniques that were used in this work. The three last chapters present the works on which this thesis can be divided. Chapter three demonstrates on free-standing films of MHPOBC (historic liquid crystal that possesses the antiferroelectric sub-phases) the possibility to extend the technique of resonant X-ray diffraction to liquid crystals without resonant element. In the fourth chapter the structure of the B 2 liquid crystal phase of bent-core molecules (or banana molecules) is elucidated by using resonant X-ray diffraction combined with polarization analysis of the diffracted beam. A model of the polarization of the resonant beam diffracted by four different structures proposed for the B 2 phase is developed in this chapter. In the fifth chapter a smectic binary mixture presenting a very original critical point of phase separation is studied by X-ray reflectivity and optical microscopy. A concentration gradient in the direction perpendicular to the plane of the film seems to be induced by the free-standing film geometry. The results of a simplified model of the system are compatible with this interpretation

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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°

  7. 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

  8. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    International Nuclear Information System (INIS)

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-01-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

  9. 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°

  10. 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°.

  11. 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

  12. 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.

  13. X-ray diffraction analysis of LiCu{sub 2}O{sub 2} crystals with additives of silver atoms

    Energy Technology Data Exchange (ETDEWEB)

    Sirotinkin, V. P., E-mail: irotinkin.vladimir@mail.ru; Bush, A. A.; Kamentsev, K. E. [Moscow State Technical University of Radio Engineering, Electronics, and Automation (Russian Federation); Dau, H. S. [People’s Friendship University of Russia (Russian Federation); Yakovlev, K. A. [Moscow State Technical University of Radio Engineering, Electronics, and Automation (Russian Federation); Tishchenko, E. A. [People’s Friendship University of Russia (Russian Federation)

    2015-09-15

    Silver-containing LiCu{sub 2}O{sub 2} crystals up to 4 × 8 × 8 mm in size were grown by the crystallization of 80(1-x)CuO · 20{sub x}AgNO{sub 3} · 20Li{sub 2}CO{sub 3} (0 ≤ x ≤ 0.5) mixture melt. According to the X-ray spectral and Rietveld X-ray diffraction data, the maximum amount of silver incorporated in the LiCu{sub 2}O{sub 2} structure is about 4 at % relative to the copper content. It was established that silver atoms occupy statistically crystallographic positions of lithium atoms. The incorporation of silver atoms is accompanied by a noticeable increase in parameter c of the LiCu{sub 2}O{sub 2} rhombic unit cell, a slight increase in parameter a, and a slight decrease in parameter b.

  14. Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Baños-Sanz, Jose Ignacio; Villate, Maider; Sanz-Aparicio, Julia; Brearley, Charles Alistair; González, Beatriz

    2009-01-01

    Inositol 1,3,4,5,6-pentakisphosphate kinase from A. thaliana has been expressed in E. coli, purified and crystallized and diffraction data have been collected to 2.3 Å resolution. Two heavy-atom crystal derivatives are under study. Inositol 1,3,4,5,6-pentakisphosphate kinase (IP 5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP 6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) and ATP. IP 6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP 6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP 5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP 5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP 6 . The best crystals obtained for X-ray crystallographic analysis belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 58.124, b = 113.591, c = 142.478 Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source

  15. 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

  16. 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

  17. Crystallization and X-ray diffraction analysis of the CH domain of the cotton kinesin GhKCH2

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Xinghua [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China); The Fourth Military Medical University, No. 169 Changlexi Road, Xincheng District, Xi’an 710032, People’s Republic of (China); Chen, Ziwei; Li, Ping; Liu, Guoqin, E-mail: liu@cau.edu.cn [China Agricultural University, No. 2 Yuanmingyuanxilu, Haidian District, Beijing 100094, People’s Republic of (China)

    2016-02-19

    The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin (K{sub d} = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P2{sub 1}, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 44.8%.

  18. 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°

  19. 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%

  20. 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

  1. 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%

  2. Crystallization and X-ray diffraction analysis of salicylate synthase, a chorismate-utilizing enyme involved in siderophore biosynthesis

    International Nuclear Information System (INIS)

    Parsons, James F.; Shi, Katherine; Calabrese, Kelly; Ladner, Jane E.

    2006-01-01

    Salicylate synthase, which catalyzes the first step in the synthesis of the siderophore yersiniabactin, has been crystallized. Diffraction data have been collected to 2.5 Å. Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence. Yersiniabactin, produced by several Enterobacteriaceae, is derived from the key metabolic intermediate chorismic acid via its conversion to salicylate by salicylate synthase. Crystals of salicylate synthase from the uropathogen Escherichia coli CFT073 have been grown by vapour diffusion using polyethylene glycol as the precipitant. The monoclinic (P2 1 ) crystals diffract to 2.5 Å. The unit-cell parameters are a = 57.27, b = 164.07, c = 59.04 Å, β = 108.8°. The solvent content of the crystals is 54% and there are two molecules of the 434-amino-acid protein in the asymmetric unit. It is anticipated that the structure will reveal key details about the reaction mechanism and the evolution of salicylate synthase

  3. Crystallization and X-ray diffraction analysis of salicylate synthase, a chorismate-utilizing enyme involved in siderophore biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Parsons, James F., E-mail: parsonsj@umbi.umd.edu; Shi, Katherine; Calabrese, Kelly [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States); Ladner, Jane E. [Center for Advanced Research in Biotechnology, The University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850 (United States); National Institute of Standards and Technology (United States)

    2006-03-01

    Salicylate synthase, which catalyzes the first step in the synthesis of the siderophore yersiniabactin, has been crystallized. Diffraction data have been collected to 2.5 Å. Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence. Yersiniabactin, produced by several Enterobacteriaceae, is derived from the key metabolic intermediate chorismic acid via its conversion to salicylate by salicylate synthase. Crystals of salicylate synthase from the uropathogen Escherichia coli CFT073 have been grown by vapour diffusion using polyethylene glycol as the precipitant. The monoclinic (P2{sub 1}) crystals diffract to 2.5 Å. The unit-cell parameters are a = 57.27, b = 164.07, c = 59.04 Å, β = 108.8°. The solvent content of the crystals is 54% and there are two molecules of the 434-amino-acid protein in the asymmetric unit. It is anticipated that the structure will reveal key details about the reaction mechanism and the evolution of salicylate synthase.

  4. 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

  5. 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

  6. 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%.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    International Nuclear Information System (INIS)

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-01-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts

  14. Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens

    International Nuclear Information System (INIS)

    Singh, Nisha; Halliday, Amy C.; Knight, Matthew; Lack, Nathan A.; Lowe, Edward; Churchill, Grant C.

    2012-01-01

    M. musculus and H. sapiens inositol monophosphatase 1 were cloned, expressed, purified and crystallized. Diffraction data were collected and analysed at resolutions of 2.4 and 1.7 Å, respectively, and the structures were compared in order to identify any structural differences. Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å

  15. 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

  16. 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.

  17. 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

  18. 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%.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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)

  4. 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

  5. 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

  6. 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

  7. 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)

  8. 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

  9. 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

  10. Multilayer optics for x-ray analysis: design - fabrication - application

    International Nuclear Information System (INIS)

    Dietsch, R.; Holz, Th.; Bruegemann, L.

    2002-01-01

    Full text: The use of multilayer optics induced a decisive extension of opportunities in laboratory based X-ray analysis. With the growing number of different applications, more and more dedicated X-ray optics are required, optimized for the spectral range they are intended to be used for. Both the characteristic of the used X-ray source and the design of the multilayer optics finally define the performance of the conditioned incident beam for the application. In any case, qualified spacer and absorber materials have to be selected for the deposition of the multilayer in respect to the designated X-ray wavelength. X-ray optical devices based on uniform multilayers have the advantage of a wide acceptance angle but show chromatic aberrations. This effect can be avoided by synthesizing a multilayer with a lateral thickness gradient. The gradient ensures that any beam of a certain wavelength emitted from an infinite narrow X-ray source impinging the multilayer optics fulfills the Bragg condition. Three different types of curvature of laterally graded multilayer mirrors are used for X-ray analysis experiments: parabolic, elliptic and planar, which result in parallel, focusing and divergent beam conditions, respectively. Furthermore, the X-ray beam characteristics: intensity, monochromasy, divergence, beam width and brilliance can be additionally conditioned by combining one multilayer optics with either a different optic and/or with a crystal monochromator. The deposition of nanometer-multilayers, used as X-ray optical components, result in extraordinary requirements of the deposition process concerning precision, reproducibility and long term stability. Across a stack of more than 150 individual layers with thicknesses in the range between 1 to 10 nm, a variation of single layer thickness considerably lower than σ D = 0.1 nm and an interface roughness below σ R = 0.25 nm have to be achieved. Thickness homogeneity Δd/d -8 have to be guaranteed across macroscopic

  11. 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.

  12. 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

  13. 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

  14. Purification, crystallization and x-ray diffraction data analysis of oxy hemoglobin-I from the catfish-Liposarcus anisitsi (Pisces)

    International Nuclear Information System (INIS)

    Smarra, A.L.S.; Arni, R.K.; Azevedo Junior, W.F. de; Colombo, M.F.; Bonilla-Rodriguez, G.O.

    1997-01-01

    Full text. Hemoglobin remains, despite the enormous amount of research involving this molecule, as a prototype of allosteric models and new conformations. The present work describes the purification crystallization and X-ray diffraction data analysis of the first hemoglobin (LHb-I) from the four components which constitutes Lopisarcus anisitsi's hemolysate. The functional behaviour of this hemoglobin has shown that proton and chloride effects are dependent on the presence of phosphates. Under these conditions emerges an alkaline Bohr effect, whereas chloride increases Hb oxygen-affinity. The usual interpretation for those findings involves pKa changes induced by phosphate binding and Cl competition for the phosphate binding site respectively. Alternatively we hypothesize that conformational changes can account for those observations. Accordingly, we have chose to perform Hb crystallization under different conditions to check for alternative conformations induced by these anions. The LHb-I has an isoelectric point of 8.1 being purified by ion-exchange chromatography on DEAE-Sephadex using a pH gradient, subsequent de ionization on amberlite M B 1 resin, concentrated and stored in liquid nitrogen until use. The protein solution was crystallized using the Sparce - matrix method, being obtained two monocrystal forms. First form: space group C 2, and cell parameters: a=185.42 A b=63.04 A c=57.59 A, α=γ= 90 deg β=92.79 deg. Crystallographic data was collected to 2.8 A. Second form: hexagonal system, a=b=63.9 A, c=327.96 A, α=β90 deg, γ=120 deg. Crystallographic data was collected to 2.7 A. The structure determination of first form has been initiated by molecular replacement methods. (author)

  15. 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.

  16. 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

  17. 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

  18. Synchrotron x-ray fluorescence and extended x-ray absorption fine structure analysis

    International Nuclear Information System (INIS)

    Chen, J.R.; Gordon, B.M.; Hanson, A.L.; Jones, K.W.; Kraner, H.W.; Chao, E.C.T.; Minkin, J.A.

    1984-01-01

    The advent of dedicated synchrotron radiation sources has led to a significant increase in activity in many areas of science dealing with the interaction of x-rays with matter. Synchrotron radiation provides intense, linearly polarized, naturally collimated, continuously tunable photon beams, which are used to determine not only the elemental composition of a complex, polyatomic, dilute material but also the chemical form of the elements with improved accuracy. Examples of the application of synchrotron radiation include experiments in synchrotron x-ray fluorescence (SXRF) analysis and extended x-ray absorption fine structure (EXAFS) analysis. New synchrotron radiation x-ray microprobes for elemental analysis in the parts per billion range are under construction at several laboratories. 76 references, 24 figures

  19. 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

  20. X-ray crystal spectroscopy of JET - a design study

    International Nuclear Information System (INIS)

    Bateman, J.E.; Hobby, M.G.; Peacock, N.J.

    1980-02-01

    This study describes the design and specification of a diagnostic system to measure the space- and time-resolved x-ray spectrum from JET discharges with high-resolution crystal spectrometers operating in the wavelength region 1 - 15A. The specification is given in terms of sensitivity, resolving power, detector, and data handling requirements, special attention being given to the problems encountered in interfacing the spectrometer arrays to the torus vacuum system and in their disposition to the machine. Shielding requirements during the active mode are evaluated and a staged diagnostic is proposed to accommodate D - T operation. (U.K.)

  1. In vivo X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Ahlgren, L.

    1980-02-01

    Measurements on five occupationally exposed persons have shown that it is possible to use X-ray fluorescence analysis for in vivo measurements of lead in the skeleton. The technique for calibrating in vivo X-ray fluorescence measurements of lead in bone tissue has been studied in detail and a two-component phantom simulating the bone and the soft tissue parts of the finger constructed. The technique has been used for in vivo measurements on 22 occupationally exposed persons. The minimum detectable concentration of lead in fingerbones was found to be around 20 μg x g -1 . The lead concentrations in their skeletons and blood were compared: the correlation was poor. The variations in lead concentrations in the skeleton have been studied in occupationally exposed persons and in samples from archaeological skeletons. The sensitivity and the minimum detectable concentration of cadmium in the kidney cortex in in vivo measurements has been studied by measurements on kidney models. The minimum detectable concentration was 20 μg x g -1 at a skin-kidney distance of 30 mm and 40 μg x g -1 at 40 mm. Five persons occupationally exposed were studied. (Author)

  2. Hybrid Powder - Single Crystal X-Ray Diffraction Instrument for Planetary Mineralogical Analysis of Unprepared Samples, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a planetary exploration XRD/XRF instrument based on a hybrid diffraction approach that complements powder XRD analysis, similar to that of the...

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Diastereoselective synthesis of ethyl ( Z)-3-(8-methylimidazo-[1,2- a]pyrid-2-yl)-2-phenylthioacrylate. X-ray crystal structure and conformational analysis

    Science.gov (United States)

    Gautier, A.; Roche, D.; Métin, J.; Carpy, A.; Madesclaire, M.

    1995-09-01

    The title compound 2, a gem vinyl sulfide ester, has been obtained diastereoselectively (de > 98%) by action of the ethyl thiophenoxyacetate carbanion on the imidazo[1,2- a]pyridinecarbaldehyde 1 in a basic medium, at low temperature. The X-ray crystal structure of 2 (C 19H 19N 2O 2S: Mr = 338.43, triclinic, P 1¯, a = 8.193(3) Å, b = 10.090(2) Å, c = 10.981(4) Å, α = 88.12(2)°, β = 78.66(4)°, γ = 78.53(2)°, V = 872.3(6) Å3, Z = 2, Dcalc = 1.29 g cm -3, λ( Mo Kα) = 0.71069 Å, μ = 0.189 mm -1, F(000) = 356, T = 293 K, R = 0.043 for 3610 observed reflections) has been determined and confirmed the Z configuration. The molecule is almost planar except for the phenyl ring situated in an approximate perpendicular plane. Despite the presence of the conjugate double bonds of the vinyl ester group (acrylate), coplanar with the imidazopyridine heterocycle, there is no evidence of π-electron delocalization over the whole structure. The crystal cohesion is ensured by a dense network of van der Waals contacts. A conformational analysis of the Z and E isomers by means of a Monte Carlo search and a stochastic dynamics simulation in CHCl 3 has shown that according to the method the Z isomer is more stable than the E isomer by about 7 to 10 kJ mol -1.

  10. Fundamentals of energy dispersive X-ray analysis

    CERN Document Server

    Russ, John C; Kiessling, R; Charles, J

    1984-01-01

    Fundamentals of Energy Dispersive X-ray Analysis provides an introduction to the fundamental principles of dispersive X-ray analysis. It presents descriptions, equations, and graphs to enable the users of these techniques to develop an intuitive and conceptual image of the physical processes involved in the generation and detection of X-rays. The book begins with a discussion of X-ray detection and measurement, which is accomplished by one of two types of X-ray spectrometer: energy dispersive or wavelength dispersive. The emphasis is on energy dispersive spectrometers, given their rather wid

  11. X-ray fluorescence method for trace analysis and imaging

    International Nuclear Information System (INIS)

    Hayakawa, Shinjiro

    2000-01-01

    X-ray fluorescence analysis has a long history as conventional bulk elemental analysis with medium sensitivity. However, with the use of synchrotron radiation x-ray fluorescence method has become a unique analytical technique which can provide tace elemental information with the spatial resolution. To obtain quantitative information of trace elemental distribution by using the x-ray fluorescence method, theoretical description of x-ray fluorescence yield is described. Moreover, methods and instruments for trace characterization with a scanning x-ray microprobe are described. (author)

  12. Femtosecond X-ray diffraction from two-dimensional protein crystals

    Directory of Open Access Journals (Sweden)

    Matthias Frank

    2014-03-01

    Full Text Available X-ray diffraction patterns from two-dimensional (2-D protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

  13. Design parameters of transmission curved crystal spectrometer for hard X-ray diagnoses

    International Nuclear Information System (INIS)

    Qian Feng; Cao Leifeng; Zhou Weimin; Zhao Zongqing; Gu Yuqiu; Yan Yonghong; Wei Lai; Xiao Shali

    2013-01-01

    The high resolving measurement of hard X-ray spectra generated in laser-produced plasma is usually performed using a cylindrically curved crystal spectrometer. In this paper, theoretical analysis and numerical simulation are performed to investigate the dependence of the energy range and resolving power on various design parameters, including crystal bending radius, source to crystal standoff distance, source size, location of the detector, etc. The investigation provides a means to design and develop cylindrically transmission curved crystal spectrometer which is used in hard X-ray diagnostics. The results show that crystal bending radius has a great influence on energy range of spectra and resolving power, and the separation between the spectral lines increases with the distance behind the focal circle faster than the line width, resulting in increased resolving power with distance. (authors)

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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%

  19. 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

  20. 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%

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Yennawar, Hemant [Pennsylvania State University, 8 Althouse Laboratory, University Park, PA 16802 (United States); Møller, Magda [Cornell High Energy Synchrotron Source, Ithaca, NY 14853 (United States); University of Copenhagen, DK-2100 Copenhagen (Denmark); Gillilan, Richard [Cornell High Energy Synchrotron Source, Ithaca, NY 14853 (United States); Yennawar, Neela, E-mail: nhy1@psu.edu [Pennsylvania State University, 8 Althouse Laboratory, University Park, PA 16802 (United States)

    2011-05-01

    The X-ray crystal structure and a small-angle X-ray scattering solution structure of sheep liver sorbitol dehydrogenase have been determined. The details of the interactions that enable the tetramer scaffold to be the functional biological unit have been analyzed. The X-ray crystal structure of sheep liver sorbitol dehydrogenase (slSDH) has been determined using the crystal structure of human sorbitol dehydrogenase (hSDH) as a molecular-replacement model. slSDH crystallized in space group I222 with one monomer in the asymmetric unit. A conserved tetramer that superposes well with that seen in hSDH (despite belonging to a different space group) and obeying the 222 crystal symmetry is seen in slSDH. An acetate molecule is bound in the active site, coordinating to the active-site zinc through a water molecule. Glycerol, a substrate of slSDH, also occupies the substrate-binding pocket together with the acetate designed by nature to fit large polyol substrates. The substrate-binding pocket is seen to be in close proximity to the tetramer interface, which explains the need for the structural integrity of the tetramer for enzyme activity. Small-angle X-ray scattering was also used to identify the quaternary structure of the tetramer of slSDH in solution.

  6. Epitaxial Ge-crystal arrays for X-ray detection

    International Nuclear Information System (INIS)

    Kreiliger, T; Falub, C V; Müller, E; Känel, H von; Isa, F; Isella, G; Chrastina, D; Bergamaschini, R; Marzegalli, A; Miglio, L; Kaufmann, R; Niedermann, P; Neels, A; Dommann, A; Meduňa, M

    2014-01-01

    Monolithic integration of an X-ray absorber layer on a Si CMOS chip might be a potentially attractive way to improve detector performance at acceptable costs. In practice this requires, however, the epitaxial growth of highly mismatched layers on a Si-substrate, both in terms of lattice parameters and thermal expansion coefficients. The generation of extended crystal defects, wafer bowing and layer cracking have so far made it impossible to put the simple concept into practice. Here we present a way in which the difficulties of fabricating very thick, defect-free epitaxial layers may be overcome. It consists of an array of densely packed, three-dimensional Ge-crystals on a patterned Si(001) substrate. The finite gap between neighboring micron-sized crystals prevents layer cracking and substrate bowing, while extended defects are driven to the crystal sidewalls. We show that the Ge-crystals are indeed defect-free, despite the lattice misfit of 4.2%. The electrical characteristics of individual Ge/Si heterojunction diodes are obtained from in-situ measurements inside a scanning electron microscope. The fabrication of monolithically integrated detectors is shown to be compatible with Si-CMOS processing

  7. 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

  8. Bent crystal X-ray optics for the diagnosis and applications of laser-produced plasmas

    International Nuclear Information System (INIS)

    Loetzsch, Robert

    2012-01-01

    method for measurement of rocking curves with high angular and spatial resolution. This technique allows to map lateral strain variations that are smaller than 10 -5 . A spatial resolution of <20 μm can be reached. In an application of this scheme, small real structure variations in strontium titanate that are connected with the material's low temperature structural phase transition, were investigated. The last part of the thesis presented an experiment in the field of spectroscopic investigations of laser-plasmas. X-ray line shape analysis of emission from ions exposed to high electric fields was carried out. An instrument was presented, that is used to record X-ray spectra with high dynamics of emission coming from laser-matter interactions at high laser intensities of up to 10 20 W/cm 2 . Such measurements are complicated by the small signal to background ratio of these experiments and is only possible by the combination of an efficient bent crystal optic with a single-photon sensitive two dimensional detector. The spectrometer allows to record the K-shell emission of silicon with a dynamics of better than 1000 and makes it possible to detect small changes in the spectrum caused by the strong electric fields of TV/m occuring in these plasmas. This unique technique allows for an probe of the highly transient atomic states of ions in these strong fields.

  9. 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

  10. 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

  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. 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

  14. 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

  15. 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%.

  16. 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%

  17. X-Ray analysis and methods for nondestructive control (On the 100-anniversary of X-ray foundation)

    International Nuclear Information System (INIS)

    Sosnin, F.R.

    1995-01-01

    Brief consideration is given to the history of X-ray discovery, formation of domestic X-ray industry. Principles of operation and potentialities of X-ray diffraction analysis, gammagraphy, radioscopy, radiometric analysis are described briefly. Domestic and foreign scientists, institutes and companies who contributed much to development of methods for nondestructive control are listed

  18. Measurement of X-ray beam emittance using crystal optics at an X-ray undulator beamline

    CERN Document Server

    Kohmura, Y; Awaji, M; Tanaka, T; Hara, T; Goto, S; Ishikawa, T

    2000-01-01

    We present a method of using crystal optics to measure the emittance of the X-ray source. Two perfect crystals set in (++) configuration work as a high-resolution collimator. The phase-space diagram (i.e. beam cross-section and angular distribution) could be determined without any assumptions on the light source. When the measurement is done at short wavelength radiation from undulator, the electron beam emittance is larger than the diffraction limit of the X-rays. Therefore, the electron beam emittance could be estimated. The measurement was done with the hard X-rays of 18.5 and 55 keV from an undulator beamline, BL 47XU, of SPring-8. The horizontal emittance of the X-ray beam was estimated to be about 7.6 nmrad, close to the designed electron beam emittance of the storage ring (7 nmrad). Some portions of the instrumental functions, such as the scattering by filters and windows along the beamline and the slight bent of the crystal planes of the monochromator, could not be precisely evaluated, but an upper li...

  19. Measurement of X-ray beam emittance using crystal optics at an X-ray undulator beamline

    International Nuclear Information System (INIS)

    Kohmura, Yoshiki; Suzuki, Yoshio; Awaji, Mitsuhiro; Tanaka, Takashi; Hara, Toru; Goto, Shunji; Ishikawa, Tetsuya

    2000-01-01

    We present a method of using crystal optics to measure the emittance of the X-ray source. Two perfect crystals set in (++) configuration work as a high-resolution collimator. The phase-space diagram (i.e. beam cross-section and angular distribution) could be determined without any assumptions on the light source. When the measurement is done at short wavelength radiation from undulator, the electron beam emittance is larger than the diffraction limit of the X-rays. Therefore, the electron beam emittance could be estimated. The measurement was done with the hard X-rays of 18.5 and 55 keV from an undulator beamline, BL 47XU, of SPring-8. The horizontal emittance of the X-ray beam was estimated to be about 7.6 nmrad, close to the designed electron beam emittance of the storage ring (7 nmrad). Some portions of the instrumental functions, such as the scattering by filters and windows along the beamline and the slight bent of the crystal planes of the monochromator, could not be precisely evaluated, but an upper limit for the vertical emittance of the electron beam could be obtained as 0.14 nmrad

  20. 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.

  1. 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

  2. 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%

  3. Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Mishra, Arjun K; Megta, Abhin Kumar; Palva, Airi; von Ossowski, Ingemar; Krishnan, Vengadesan

    2017-06-01

    SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Å were eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Å resolution.

  4. 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

  5. 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

  6. Effect of Electric Voltage and Current of X-ray Chamber on the Element inthe Zirconium Alloy Analysis X-ray by X-ray Fluorescence

    International Nuclear Information System (INIS)

    Yusuf-Nampira; Narko-Wibowo, L; Rosika-Krisnawati; Nudia-Barenzani

    2000-01-01

    The using of x-ray fluorescence in the chemical analysis depend heavilyon the parameters of x-ray chamber, for examples : electric voltage andelectric current. That parameter give effect in the result of determine ofSn, Cr, Fe and Ni in the zirconium alloy. 20 kV electric voltages are used onthe Mo x-ray chamber shall product x-ray of zirconium in the sample materialcan give effect in the stability of the analysis result (deviation more than5%). The result of analysis of elements in the zirconium alloy shall givedeviation less than 5% when using of electric voltage of the x-ray chamberless than 19 kV. The sensitivity of analysis can be reached by step upelectric current of x-ray chamber. (author)

  7. X-ray diagnostics for TFTR

    International Nuclear Information System (INIS)

    von Goeler, S.; Hill, K.W.; Bitter, M.

    1982-12-01

    A short description of the x-ray diagnostic preparation for the TFTR tokamak is given. The x-ray equipment consists of the limiter x-ray monitoring system, the soft x-ray pulse-height-analysis-system, the soft x-ray imaging system and the x-ray crystal spectrometer. Particular attention is given to the radiation protection of the x-ray systems from the neutron environment

  8. X-ray analysis in lung leptospira disease

    International Nuclear Information System (INIS)

    Deng Shiyong; Peng Shi; He Guoman

    2006-01-01

    Objective: To analysis the X-ray signs and subtype of the lung leptospira disease, and improve the undersdand, reduce the error diagnosis of this diseases. Methods: 40 cases of lung leptospira disease were evaluated about the check X-ray sings and clinical data, the check X-ray sings were dynamic observated and typed, and 40 cases had a diagnostic treatment. Results: There were various X-ray changes of lung leptospira disease. in 40 cases, 12 cases (30%) pulmonary marking, 21 cases (52%) little lesions, and 7 cases(18%) lager lesions, respectively. The patients who were correctly diagnosed made a recovery after effective treatment, the patients who were error diagnosed died because of multiple system organ damage. Conclusion: The check X-ray signs in lung leptospira disease have some characteristics. It may play an important role in improving this disease' diagnosis combining the dynamic observation of check X-ray sings with clinical data. (authors)

  9. 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.

  10. 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

  11. 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%.

  12. 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%

  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. 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

  16. 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.

  17. Quantitative analysis with energy dispersive X-ray fluorescence analyser

    International Nuclear Information System (INIS)

    Kataria, S.K.; Kapoor, S.S.; Lal, M.; Rao, B.V.N.

    1977-01-01

    Quantitative analysis of samples using radioisotope excited energy dispersive x-ray fluorescence system is described. The complete set-up is built around a locally made Si(Li) detector x-ray spectrometer with an energy resolution of 220 eV at 5.94 KeV. The photopeaks observed in the x-ray fluorescence spectra are fitted with a Gaussian function and the intensities of the characteristic x-ray lines are extracted, which in turn are used for calculating the elemental concentrations. The results for a few typical cases are presented. (author)

  18. Project Title: Radiochemical Analysis by High Sensitivity Dual-Optic Micro X-ray Fluorescence

    International Nuclear Information System (INIS)

    Havrilla, George J.; Gao, Ning

    2002-01-01

    A novel dual-optic micro X-ray fluorescence instrument will be developed to do radiochemical analysis of high-level radioactive wastes at DOE sites such as Savannah River Site and Hanford. This concept incorporates new X-ray optical elements such as monolithic polycapillaries and double bent crystals, which focus X-rays. The polycapillary optic can be used to focus X-rays emitted by the X-ray tube thereby increasing the X-ray flux on the sample over 1000 times. Polycapillaries will also be used to collect the X-rays from the excitation site and screen the radiation background from the radioactive species in the specimen. This dual-optic approach significantly reduces the background and increases the analyte signal thereby increasing the sensitivity of the analysis. A doubly bent crystal used as the focusing optic produces focused monochromatic X-ray excitation, which eliminates the bremsstrahlung background from the X-ray source. The coupling of the doubly bent crystal for monochromatic excitation with a polycapillary for signal collection can effectively eliminate the noise background and radiation background from the specimen. The integration of these X-ray optics increases the signal-to-noise and thereby increases the sensitivity of the analysis for low-level analytes. This work will address a key need for radiochemical analysis of high-level waste using a non-destructive, multi-element, and rapid method in a radiation environment. There is significant potential that this instrumentation could be capable of on-line analysis for process waste stream characterization at DOE sites

  19. 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

  20. 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.

  1. Structural Characterization of Doped GaSb Single Crystals by X-ray Topography

    Energy Technology Data Exchange (ETDEWEB)

    Honnicke, M.G.; Mazzaro, I.; Manica, J.; Benine, E.; M da Costa, E.; Dedavid, B. A.; Cusatis, C.; Huang, X. R.

    2009-09-13

    We characterized GaSb single crystals containing different dopants (Al, Cd and Te), grown by the Czochralski method, by x-ray topography and high angular resolution x-ray diffraction. Lang topography revealed dislocations parallel and perpendicular to the crystal's surface. Double-crystal GaSb 333 x-ray topography shows dislocations and vertical stripes than can be associated with circular growth bands. We compared our high-angular resolution x-ray diffraction measurements (rocking curves) with the findings predicted by the dynamical theory of x-ray diffraction. These measurements show that our GaSb single crystals have a relative variation in the lattice parameter ({Delta}d/d) on the order of 10{sup -5}. This means that they can be used as electronic devices (detectors, for example) and as x-ray monochromators.

  2. Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L.

    Science.gov (United States)

    Ehmke, Veronika; Winkler, Edwin; Banner, David W; Haap, Wolfgang; Schweizer, W Bernd; Rottmann, Matthias; Kaiser, Marcel; Freymond, Céline; Schirmeister, Tanja; Diederich, François

    2013-06-01

    The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition constants in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed

  3. 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

  4. 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

  5. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Chad R. [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States); Hao, Quan [MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853-8001 (United States); Stipanuk, Martha H., E-mail: mhs6@cornell.edu [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States)

    2005-11-01

    Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  6. A novel noncovalent complex of chorismate mutase and DAHP synthase from Mycobacterium tuberculosis: protein purification, crystallization and X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Ökvist, Mats; Sasso, Severin; Roderer, Kathrin; Kast, Peter; Krengel, Ute

    2009-01-01

    Two shikimate-pathway enzymes from M. tuberculosis, the intracellular chorismate mutase (MtCM) and DAHP synthase (MtDS), were produced recombinantly and purified. MtCM was crystallized alone and in complex with MtDS and analyzed by X-ray diffraction. Chorismate mutase catalyzes a key step in the shikimate-biosynthetic pathway and hence is an essential enzyme in bacteria, plants and fungi. Mycobacterium tuberculosis contains two chorismate mutases, a secreted and an intracellular one, the latter of which (MtCM; Rv0948c; 90 amino-acid residues; 10 kDa) is the subject of this work. Here are reported the gene expression, purification and crystallization of MtCM alone and of its complex with another shikimate-pathway enzyme, DAHP synthase (MtDS; Rv2178c; 472 amino-acid residues; 52 kDa), which has been shown to enhance the catalytic efficiency of MtCM. The MtCM–MtDS complex represents the first noncovalent enzyme complex from the common shikimate pathway to be structurally characterized. Soaking experiments with a transition-state analogue are also reported. The crystals of MtCM and the MtCM–MtDS complex diffracted to 1.6 and 2.1 Å resolution, respectively

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. Quantitative X ray analysis system. User's manual and guide to X ray fluorescence technique

    International Nuclear Information System (INIS)

    2009-01-01

    This guide covers trimmed and re-arranged version 3.6 of the Quantitative X ray Analysis System (QXAS) software package that includes the most frequently used methods of quantitative analysis. QXAS is a comprehensive quantitative analysis package that has been developed by the IAEA through research and technical contracts. Additional development has also been carried out in the IAEA Laboratories in Seibersdorf where QXAS was extensively tested. New in this version of the manual are the descriptions of the Voigt-profile peak fitting, the backscatter fundamental parameters' and emission-transmission methods of chemical composition analysis, an expanded chapter on the X ray fluorescence physics, and completely revised and increased number of practical examples of utilization of the QXAS software package. The analytical data accompanying this manual were collected in the IAEA Seibersdorf Laboratories in the years 2006/2007

  14. The X-ray crystal bichromator possible modifications and applications

    Czech Academy of Sciences Publication Activity Database

    Hrdý, Jaromír

    2017-01-01

    Roč. 854, May (2017), s. 1-2 ISSN 0168-9002 Institutional support: RVO:68378271 Keywords : X-ray bichromator * X-ray optics * synchrotron radiation optics Subject RIV: BM - Solid Matter Physics ; Magnetism OBOR OECD: Condensed matter physics (including formerly solid state physics, supercond.) Impact factor: 1.362, year: 2016

  15. Understanding Nickel Thin Film crystallization using X-Ray ...

    African Journals Online (AJOL)

    The microstructures of these Ni films were studied using X-ray diffractometry technique. The X-ray diffraction (XRD) patterns depicted 100% and 42% relative intensity (RI) peaks identified for normal and helical deposited Ni films but none for the zigzag deposited Ni film. Higher degree of crystallinity of Ni was demonstrated ...

  16. Crystal Structures and Small-angle X-ray Scattering Analysis of UDP-galactopyranose Mutase from the Pathogenic Fungus Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Dhatwalia, Richa; Singh, Harkewal; Oppenheimer, Michelle; Karr, Dale B.; Nix, Jay C.; Sobrado, Pablo; Tanner, John J. (LBNL); (Missouri); (VPI)

    2015-10-15

    UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 {angstrom} to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.

  17. Nanosecond X-ray detector based on high resistivity ZnO single crystal semiconductor

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xiaolong; He, Yongning, E-mail: yongning@mail.xjtu.edu.cn; Peng, Wenbo; Huang, Zhiyong; Qi, Xiaomeng; Pan, Zijian; Zhang, Wenting [School of Electronic and Information Engineering, Xi' an Jiaotong University, Xi' an 710049 (China); Chen, Liang; Liu, Jinliang; Zhang, Zhongbing; Ouyang, Xiaoping [Radiation Detection Research Center, Northwest Institute of Nuclear Technology, Xi' an 710024 (China)

    2016-04-25

    The pulse radiation detectors are sorely needed in the fields of nuclear reaction monitoring, material analysis, astronomy study, spacecraft navigation, and space communication. In this work, we demonstrate a nanosecond X-ray detector based on ZnO single crystal semiconductor, which emerges as a promising compound-semiconductor radiation detection material for its high radiation tolerance and advanced large-size bulk crystal growth technique. The resistivity of the ZnO single crystal is as high as 10{sup 13} Ω cm due to the compensation of the donor defects (V{sub O}) and acceptor defects (V{sub Zn} and O{sub i}) after high temperature annealing in oxygen. The photoconductive X-ray detector was fabricated using the high resistivity ZnO single crystal. The rise time and fall time of the detector to a 10 ps pulse electron beam are 0.8 ns and 3.3 ns, respectively, indicating great potential for ultrafast X-ray detection applications.

  18. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands.

    Science.gov (United States)

    Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta

    2011-02-01

    Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

  19. X-ray spectrometry and X-ray microtomography techniques for soil and geological samples analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kubala-Kukuś, A.; Banaś, D.; Braziewicz, J. [Institute of Physics, Jan Kochanowski University, ul. Świetokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Artwińskiego 3, 25-734 Kielce (Poland); Dziadowicz, M.; Kopeć, E. [Institute of Physics, Jan Kochanowski University, ul. Świetokrzyska 15, 25-406 Kielce (Poland); Majewska, U. [Institute of Physics, Jan Kochanowski University, ul. Świetokrzyska 15, 25-406 Kielce (Poland); Holycross Cancer Center, ul. Artwińskiego 3, 25-734 Kielce (Poland); Mazurek, M.; Pajek, M.; Sobisz, M.; Stabrawa, I. [Institute of Physics, Jan Kochanowski University, ul. Świetokrzyska 15, 25-406 Kielce (Poland); Wudarczyk-Moćko, J. [Holycross Cancer Center, ul. Artwińskiego 3, 25-734 Kielce (Poland); Góźdź, S. [Holycross Cancer Center, ul. Artwińskiego 3, 25-734 Kielce (Poland); Institute of Public Health, Jan Kochanowski University, IX Wieków Kielc 19, 25-317 Kielce (Poland)

    2015-12-01

    A particular subject of X-ray fluorescence analysis is its application in studies of the multielemental sample of composition in a wide range of concentrations, samples with different matrices, also inhomogeneous ones and those characterized with different grain size. Typical examples of these kinds of samples are soil or geological samples for which XRF elemental analysis may be difficult due to XRF disturbing effects. In this paper the WDXRF technique was applied in elemental analysis concerning different soil and geological samples (therapeutic mud, floral soil, brown soil, sandy soil, calcium aluminum cement). The sample morphology was analyzed using X-ray microtomography technique. The paper discusses the differences between the composition of samples, the influence of procedures with respect to the preparation of samples as regards their morphology and, finally, a quantitative analysis. The results of the studies were statistically tested (one-way ANOVA and correlation coefficients). For lead concentration determination in samples of sandy soil and cement-like matrix, the WDXRF spectrometer calibration was performed. The elemental analysis of the samples was complemented with knowledge of chemical composition obtained by X-ray powder diffraction.

  20. X-ray spectrometry and X-ray microtomography techniques for soil and geological samples analysis

    International Nuclear Information System (INIS)

    Kubala-Kukuś, A.; Banaś, D.; Braziewicz, J.; Dziadowicz, M.; Kopeć, E.; Majewska, U.; Mazurek, M.; Pajek, M.; Sobisz, M.; Stabrawa, I.; Wudarczyk-Moćko, J.; Góźdź, S.

    2015-01-01

    A particular subject of X-ray fluorescence analysis is its application in studies of the multielemental sample of composition in a wide range of concentrations, samples with different matrices, also inhomogeneous ones and those characterized with different grain size. Typical examples of these kinds of samples are soil or geological samples for which XRF elemental analysis may be difficult due to XRF disturbing effects. In this paper the WDXRF technique was applied in elemental analysis concerning different soil and geological samples (therapeutic mud, floral soil, brown soil, sandy soil, calcium aluminum cement). The sample morphology was analyzed using X-ray microtomography technique. The paper discusses the differences between the composition of samples, the influence of procedures with respect to the preparation of samples as regards their morphology and, finally, a quantitative analysis. The results of the studies were statistically tested (one-way ANOVA and correlation coefficients). For lead concentration determination in samples of sandy soil and cement-like matrix, the WDXRF spectrometer calibration was performed. The elemental analysis of the samples was complemented with knowledge of chemical composition obtained by X-ray powder diffraction.

  1. Time dependence of X-ray polarizability of a crystal induced by an intense femtosecond X-ray pulse

    Directory of Open Access Journals (Sweden)

    A. Leonov

    2014-11-01

    Full Text Available The time evolution of the electron density and the resulting time dependence of Fourier components of the X-ray polarizability of a crystal irradiated by highly intense femtosecond pulses of an X-ray free-electron laser (XFEL is investigated theoretically on the basis of rate equations for bound electrons and the Boltzmann equation for the kinetics of the unbound electron gas. The photoionization, Auger process, electron-impact ionization, electron–electron scattering and three-body recombination have been implemented in the system of rate equations. An algorithm for the numerical solution of the rate equations was simplified by incorporating analytical expressions for the cross sections of all the electron configurations in ions within the framework of the effective charge model. Using this approach, the time dependence of the inner shell populations during the time of XFEL pulse propagation through the crystal was evaluated for photon energies between 4 and 12 keV and a pulse width of 40 fs considering a flux of 1012 photons pulse−1 (focusing on a spot size of ∼1 µm. This flux corresponds to a fluence ranging between 0.8 and 2.4 mJ µm−2. The time evolution of the X-ray polarizability caused by the change of the atomic scattering factor during the pulse propagation is numerically analyzed for the case of a silicon crystal. The time-integrated polarizability drops dramatically if the fluence of the X-ray pulse exceeds 1.6 mJ µm−2.

  2. Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNAIle2 and ATP

    International Nuclear Information System (INIS)

    Osawa, Takuo; Inanaga, Hideko; Kimura, Satoshi; Terasaka, Naohiro; Suzuki, Tsutomu; Numata, Tomoyuki

    2011-01-01

    A. fulgidus TiaS was cocrystallized with tRNA Ile2 and ATP and X-ray diffraction data were collected to 2.9 Å resolution using a synchrotron-radiation source. The cytidine at the first anticodon position of archaeal tRNA Ile2 , which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm 2 C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA Ile -agm 2 C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA Ile2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA Ile2 and ATP by the vapour-diffusion method. The crystals of the TiaS–tRNA Ile2 –ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3 2 21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNA Ile2 –ATP complex, with a Matthews coefficient of 2.8 Å 3 Da −1 and a solvent content of 61%

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the IL-20-IL-20R1-IL-20R2 complex

    Energy Technology Data Exchange (ETDEWEB)

    Logsdon, Naomi J.; Allen, Christopher E.; Rajashankar, Kanagalaghatta R.; Walter, Mark R. (Cornell); (UAB)

    2012-02-08

    Interleukin-20 (IL-20) is an IL-10-family cytokine that regulates innate and adaptive immunity in skin and other tissues. In addition to protecting the host from various external pathogens, dysregulated IL-20 signaling has been shown to contribute to the pathogenesis of human psoriasis. IL-20 signals through two cell-surface receptor heterodimers, IL-20R1-IL-20R2 and IL-22R1-IL-20R2. In this report, crystals of the IL-20-IL-20R1-IL-20R2 ternary complex have been grown from polyethylene glycol solutions. The crystals belonged to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = 111, c = 135 {angstrom}, and diffracted X-rays to 3 {angstrom} resolution. The crystallographic asymmetric unit contains one IL-20-IL-20R1-IL-20R2 complex, corresponding to a solvent content of approximately 54%.

  4. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation

    International Nuclear Information System (INIS)

    Barbosa, Rosicler Lázaro; Rinaldi, Fábio Cupri; Guimarães, Beatriz Gomes; Benedetti, Celso Eduardo

    2007-01-01

    In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit

  5. 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

  6. Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

    International Nuclear Information System (INIS)

    Asojo, Oluwatoyin A.; Loukas, Alex; Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M.; Hotez, Peter J.

    2005-01-01

    In order to clarify the structural basis of the pathogenesis-related-1 domain, Na-ASP-1, the first multi-domain ASP from the human hookworm parasite N. americanus, has been crystallized. 2.2 Å resolution data have been collected from a crystal belonging to the monoclinic space group P2 1 . Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P2 1 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit

  7. Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [Eppley Institute for Research in Cancer and Allied Diseases, 987696 Nebraska Medical Center, Omaha, NE 68198-7696 (United States); Loukas, Alex [Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Washington DC 20037 (United States); Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, QLD 4006 (Australia); Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M. [Department of Chemical Engineering, The University of Nebraska-Lincoln, Lincoln, NE 68588-0643 (United States); Hotez, Peter J. [Department of Microbiology and Tropical Medicine, The George Washington University Medical Center, Washington DC 20037 (United States); Eppley Institute for Research in Cancer and Allied Diseases, 987696 Nebraska Medical Center, Omaha, NE 68198-7696 (United States)

    2005-04-01

    In order to clarify the structural basis of the pathogenesis-related-1 domain, Na-ASP-1, the first multi-domain ASP from the human hookworm parasite N. americanus, has been crystallized. 2.2 Å resolution data have been collected from a crystal belonging to the monoclinic space group P2{sub 1}. Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P2{sub 1} with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit.

  8. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Barbosa, Rosicler Lázaro; Rinaldi, Fábio Cupri; Guimarães, Beatriz Gomes, E-mail: beatriz@lnls.br; Benedetti, Celso Eduardo, E-mail: beatriz@lnls.br [Center for Molecular and Structural Biology, Brazilian Synchrotron Light Laboratory, Campinas, SP, CP 6192, CEP 13083-970 (Brazil)

    2007-07-01

    In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.

  9. Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Jung-Sue [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of); Rhee, Jin-Kyu [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Dong-Uk [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Oh, Jong-Won [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Protein Network Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2006-02-01

    Recombinant EstE1 protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was then crystallized at 290 K by the hanging-drop vapour-diffusion method. EstE1, a new thermostable esterase, was isolated by functional screening of a metagenomic DNA library from thermal environment samples. This enzyme showed activity towards short-chain acyl derivatives of length C4–C6 at a temperature of 303–363 K and displayed a high thermostability above 353 K. EstE1 has 64 and 57% amino-acid sequence similarity to est{sub pc}-encoded carboxylesterase from Pyrobaculum calidifontis and AFEST from Archaeoglobus fulgidus, respectively. The recombinant protein with a histidine tag at the C-terminus was overexpressed in Escherichia coli strain BL21(DE3) and then purified by affinity chromatography. The protein was crystallized at 290 K by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.3 Å resolution from an EstE1 crystal; the crystal belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 73.71, c = 234.23 Å. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient V{sub M} is calculated to be 2.2 Å{sup 3} Da{sup −1} and the solvent content is 44.1%.

  10. Non-covalent interactions in 2-methylimidazolium copper(II) complex (MeImH)2[Cu(pfbz)4]: Synthesis, characterization, single crystal X-ray structure and packing analysis

    Science.gov (United States)

    Sharma, Raj Pal; Saini, Anju; Kumar, Santosh; Kumar, Jitendra; Sathishkumar, Ranganathan; Venugopalan, Paloth

    2017-01-01

    A new anionic copper(II) complex, (MeImH)2 [Cu(pfbz)4] (1) where, MeImH = 2-methylimidazolium and pfbz = pentafluorobenzoate has been isolated by reacting copper(II) sulfate pentahydrate, pentafluorobenzoic acid and 2-methylimidazole in ethanol: water mixture in 1:2:2 molar ratio. This complex 1 has been characterized by elemental analysis, thermogravimetric analysis, spectroscopic techniques (UV-Vis, FT-IR) and conductance measurements. The complex salt crystallizes in monoclinic crystal system with space group C2/c. Single crystal X-ray structure determination revealed the presence of discrete ions: [Cu(pfbz)4]2- anion and two 2-methylimidazolium cation (C4H7N2)+. The crystal lattice is stabilized by strong hydrogen bonding and F⋯F interactions between cationic-anionic and the anionic-anionic moieties respectively, besides π-π interactions.

  11. Crystallization, X-ray diffraction analysis and SIRAS/molecular-replacenent phasing of three crystal forms of Anabaena sensory rhodopsin transducer

    International Nuclear Information System (INIS)

    Vogeley, Lutz; Luecke, Hartmut

    2006-01-01

    Crystals of Anabaena sensory rhodopsin transducer, the transducer for the cyanobacterial photosensor Anabaena sensory rhodopsin, obtained in the space groups P4, C2 and P2 1 2 1 2 1 diffract to 1.8, 2.1 and 2.0 Å, respectively. Phases for these crystal forms were obtained by SIRAS phasing using an iodide quick-soak derivative (P4) and molecular replacement (C2 and P2 1 2 1 2 1 ). Anabaena sensory rhodopsin transducer (ASRT) is a 14.7 kDa soluble signaling protein associated with the membrane-embedded light receptor Anabaena sensory rhodopsin (ASR) from Anabaena sp., a freshwater cyanobacterium. Crystals of ASRT were obtained in three different space groups, P4, C2 and P2 1 2 1 2 1 , which diffract to 1.8, 2.1 and 2.0 Å, respectively. Phases for one of these crystal forms (P4) were obtained by SIRAS phasing using an iodide quick-soak derivative and a partial model was built. Phases for the remaining crystal forms were obtained by molecular replacement using the partial model from the P4 crystal form

  12. 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.

  13. Limestone rocks analysis by X-ray fluorescence analysis

    International Nuclear Information System (INIS)

    Izquierdo M, G.; Ponce R, R.; Vazquez J, J.

    1996-01-01

    By request of a private company, employing basically X-ray fluorescence analysis (X RF), was established a fast and accurate method for the analysis of the major elements in limestone rocks. Additionally, for complementing analysis was determined by ion chromatography, the chlorides appearance and by atomic absorption of sodium. By gravimetry, was determined the losses by ignition and the alpha quartz. (Author)

  14. Tissue chemical analysis with muonic X-rays

    International Nuclear Information System (INIS)

    Hutson, R.L.; Reidy, J.J.; Springer, K.; Daniel, H.; Knowles, H.B.

    1976-01-01

    The stopped muon channel at the Clinton P. Anderson Meson Physics Facility (LAMPF) was used as a source of muons for studying the elemental composition of tissue with muonic X rays. The X ray spectra from several types of tissue were used to determine the amounts of carbon, nitrogen, and oxygen present. These determinations agree with the results of more conventional chemical analysis. The results show that muonic X rays offer a non-invasive technique for determining the amounts of the more abundant elements present in selected regions of the body. (orig.) [de

  15. Local layer structure of smectic liquid crystals by X-ray micro-diffraction

    CERN Document Server

    Takanishi, Y

    2003-01-01

    The local layer structure of smectic liquid crystal has been measured using time-resolved synchrotron X-ray micro-diffraction. Typical layer disorders observed in surface stabilized (anti-) ferroelectric liquid crystals, i.e. a stripe texture, a needed-