Equilibrium Kinetics Studies and Crystallization Aboard the International Space Station (ISS) Using the Protein Crystallization Apparatus for Microgravity (PCAM)

Science.gov (United States)

Achari, Aniruddha; Roeber, Dana F.; Barnes, Cindy L.; Kundrot, Craig E.; Stinson, Thomas N. (Technical Monitor)

2002-01-01

Protein Crystallization Apparatus in Microgravity (PCAM) trays have been used in Shuttle missions to crystallize proteins in a microgravity environment. The crystallization experiments are 'sitting drops' similar to that in Cryschem trays, but the reservoir solution is soaked in a wick. From early 2001, crystallization experiments are conducted on the International Space Station using mission durations of months rather than two weeks on previous shuttle missions. Experiments were set up in April 2001 on Flight 6A to characterize the time crystallization experiments will take to reach equilibrium in a microgravity environment using salts, polyethylene glycols and an organic solvent as precipitants. The experiments were set up to gather data for a series of days of activation with different droplet volumes and precipitants. The experimental set up on ISS and results of this study will be presented. These results will help future users of PCAM to choose precipitants to optimize crystallization conditions for their target macromolecules for a particular mission with known mission duration. Changes in crystal morphology and size between the ground and space grown crystals of a protein and a protein -DNA complex flown on the same mission will also be presented.

Split green fluorescent protein as a modular binding partner for protein crystallization

International Nuclear Information System (INIS)

Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

2013-01-01

A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

Protein crystal growth in low gravity

Science.gov (United States)

Feigelson, Robert S.

1993-01-01

This Final Technical Report for NASA Grant NAG8-774 covers the period from April 27, 1989 through December 31, 1992. It covers five main topics: fluid flow studies, the influence of growth conditions on the morphology of isocitrate lyase crystals, control of nucleation, the growth of lysozyme by the temperature gradient method and graphoepitaxy of protein crystals. The section on fluid flow discusses the limits of detectability in the Schlieren imaging of fluid flows around protein crystals. The isocitrate lyase study compares crystals grown terrestrially under a variety of conditions with those grown in space. The controlling factor governing the morphology of the crystals is the supersaturation. The lack of flow in the interface between the drop and the atmosphere in microgravity causes protein precipitation in the boundary layer and a lowering of the supersaturation in the drop. This lowered supersaturation leads to improved crystal morphology. Preliminary experiments with lysozyme indicated that localized temperature gradients could be used to nucleate crystals in a controlled manner. An apparatus (thermonucleator) was designed to study the controlled nucleation of protein crystals. This apparatus has been used to nucleate crystals of materials with both normal (ice-water, Rochelle salt and lysozyme) and retrograde (horse serum albumin and alpha chymotrypsinogen A) solubility. These studies have lead to the design of an new apparatus that small and more compatible with use in microgravity. Lysozyme crystals were grown by transporting nutrient from a source (lysozyme powder) to the crystal in a temperature gradient. The influence of path length and cross section on the growth rate was demonstrated. This technique can be combined with the thermonucleator to control both nucleation and growth. Graphoepitaxy utilizes a patterned substrate to orient growing crystals. In this study, silicon substrates with 10 micron grooves were used to grow crystals of catalase

Protein Crystals as Novel Catalytic Materials.

Science.gov (United States)

Margolin, Alexey L.; Navia, Manuel A.

2001-06-18

In this era of molecular biology, protein crystallization is often considered to be a necessary first step in obtaining structural information through X-ray diffraction analysis. In a different light, protein crystals can also be thought of as materials, whose chemical and physical properties make them broadly attractive and useful across a larger spectrum of disciplines. The full potential of these protein crystalline materials has been severely restricted in practice, however, both by their inherent fragility, and by strongly held skepticism concerning their routine and predictable growth, formulation, and practical application. Fortunately, these problems have turned out to be solvable. A systematic exploration of the biophysics and biochemistry of protein crystallization has shown that one can dependably create new protein crystalline materials more or less at will. In turn, these crystals can be readily strengthened, both chemically and mechanically, to make them suitable for practical commercialization. Today, these novel materials are used as industrial catalysts on a commercial scale, in bioremediation and "green chemistry" applications, and in enantioselective chromatography of pharmaceuticals and fine chemicals. In the near future, their utility will expand, to include the purification of protein drugs, formulation of direct protein therapeutics, and development of adjuvant-less vaccines.

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

International Nuclear Information System (INIS)

Gerdts, Cory J.; Elliott, Mark; Lovell, Scott; Mixon, Mark B.; Napuli, Alberto J.; Staker, Bart L.; Nollert, Peter; Stewart, Lance

2008-01-01

The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase. The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ‘hybrid’ crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants

Expression, purification and crystallization of the Cmi immunity protein from Escherichia coli

International Nuclear Information System (INIS)

Römer, Christin; Patzer, Silke I.; Albrecht, Reinhard; Zeth, Kornelius; Braun, Volkmar

2011-01-01

The colicin M immunity protein Cmi protects E. coli cells against killing by colicin M. The Cmi protein was produced for structure determination and crystals were obtained which diffracted to high resolution. Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 Å in the orthorhombic space group C222 1 . The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 Å, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%

Analysis of crystallization data in the Protein Data Bank

International Nuclear Information System (INIS)

Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

2015-01-01

In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored

Analysis of crystallization data in the Protein Data Bank

Energy Technology Data Exchange (ETDEWEB)

Kirkwood, Jobie [University of York, York YO10 5DD (United Kingdom); Hargreaves, David [AstraZeneca, Darwin Building, Cambridge Science Park, Cambridge CB4 0WG (United Kingdom); O’Keefe, Simon [University of York, York YO10 5DD (United Kingdom); Wilson, Julie, E-mail: julie.wilson@york.ac.uk [University of York, York YO10 5DD (United Kingdom); University of York, York YO10 5DD (United Kingdom)

2015-09-23

In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

KAUST Repository

Dumbrack, Roland

2016-01-26

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

Directory of Open Access Journals (Sweden)

Alves Lúcia M. Carareto

2000-01-01

Full Text Available The development of the production and use of Bacillus thuringiensis in Brazil at a commercial scale faces certain difficulties, among them the establishment of efficient methodologies for the quantitation of toxic products to be commercialized. Presently, the amount of toxin is given in percentage by analyzing the samples total protein content. Such methodology however, does not measure the actual amount of active protein present in the product, since most strains express different endotoxin genes and might even produce b-toxin. Since the various types of toxins exhibit different antigenic characteristics, this work has as objective the utilization of fast immunological techniques to quantify the level of crystal protein. Crystal protein produced by a subspecies of Bacillus thuringiensis var. israelensis was purified by ultracentrifugation and utilized to immunize rabbits and to produce hiperimmune sera. Such sera were latter used to evaluate the level of proteins on commercial bioinsecticide and on laboratory cultures of B. thuringiensis through the immunodot technique. The results were obtained by comparison of data obtained from reactions with known concentrations of crystal protein permitting to evaluate the level of such protein on various materials.

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.

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

Analysis of crystallization data in the Protein Data Bank.

Science.gov (United States)

Kirkwood, Jobie; Hargreaves, David; O'Keefe, Simon; Wilson, Julie

2015-10-01

The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

Ultratight crystal packing of a 10 kDa protein

Energy Technology Data Exchange (ETDEWEB)

Trillo-Muyo, Sergio [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain); Jasilionis, Andrius [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Domagalski, Marcin J. [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Chruszcz, Maksymilian [University of South Carolina, 631 Sumter Street, Columbia, SC 29208 (United States); Minor, Wladek [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Kuisiene, Nomeda [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Arolas, Joan L.; Solà, Maria; Gomis-Rüth, F. Xavier, E-mail: xgrcri@ibmb.csic.es [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain)

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Directory of Open Access Journals (Sweden)

Sergio Orduz

1996-04-01

Full Text Available Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt subsp. medellin (Btmed was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm. In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200. Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed

Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

Science.gov (United States)

Höfte, Herman; Van Rie, Jeroen; Jansens, Stefan; Van Houtven, Annemie; Vanderbruggen, Hilde; Vaeck, Mark

1988-01-01

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity. Images PMID:16347711

The contribution of polystyrene nanospheres towards the crystallization of proteins.

Directory of Open Access Journals (Sweden)

Johanna M Kallio

Full Text Available BACKGROUND: Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins. METHODOLOGY: The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT, and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times. CONCLUSIONS: All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.

Development of an X-ray fluorescence holographic measurement system for protein crystals

International Nuclear Information System (INIS)

Sato-Tomita, Ayana; Shibayama, Naoya; Okabe, Takahiro; Happo, Naohisa; Kimura, Koji; Matsushita, Tomohiro; Park, Sam-Yong; Sasaki, Yuji C.; Hayashi, Kouichi

2016-01-01

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α_2β_2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm"3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Structure of a second crystal form of Bence-Jones protein Loc: Strikingly different domain associations in two crystal forms of a single protein

International Nuclear Information System (INIS)

Schiffer, M.; Ainsworth, C.; Xu, Z.B.; Carperos, W.; Olsen, K.; Solomon, A.; Stevens, F.J.; Chang, C.H.

1989-01-01

The authors have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-angstrom resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding pocket. The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion. The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry

Microphase Separation Controlled beta-Sheet Crystallization Kinetics in Fibrous Proteins

International Nuclear Information System (INIS)

Hu, X.; Lu, Q.; Kaplan, D.; Cebe, P.

2009-01-01

Silk is a naturally occurring fibrous protein with a multiblock chain architecture. As such, it has many similarities with synthetic block copolymers, including the possibility for e-sheet crystallization restricted within the crystallizable blocks. The mechanism of isothermal crystallization kinetics of e-sheet crystals in silk multiblock fibrous proteins is reported in this study. Kinetics theories, such as Avrami analysis which was established for studies of synthetic polymer crystal growth, are for the first time extended to investigate protein self-assembly in e-sheet rich Bombyx mori silk fibroin samples, using time-resolved Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and synchrotron real-time wide-angle X-ray scattering (WAXS). The Avrami exponent, n, was close to 2 for all methods and crystallization temperatures, indicating formation of e-sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in synthetic polymers. Observations by scanning electron microscopy support the view that the protein structures vary during the different stages of crystal growth, and show a microphase separation pattern after chymotrypsin enzyme biodegradation. We present a model to explain the crystallization of the multiblock silk fibroin protein, by analogy to block copolymers: crystallization of e-sheets occurs under conditions of geometrical restriction caused by phase separation of the crystallizable and uncrystallizable blocks. This crystallization model could be widely applicable in other proteins with multiblock (i.e., crystallizable and noncrystallizable) domains.

Automating the application of smart materials for protein crystallization

International Nuclear Information System (INIS)

Khurshid, Sahir; Govada, Lata; EL-Sharif, Hazim F.; Reddy, Subrayal M.; Chayen, Naomi E.

2015-01-01

The first semi-liquid, non-protein nucleating agent for automated protein crystallization trials is described. This ‘smart material’ is demonstrated to induce crystal growth and will provide a simple, cost-effective tool for scientists in academia and industry. The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as ‘smart materials’) for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials

Development of an X-ray fluorescence holographic measurement system for protein crystals

Energy Technology Data Exchange (ETDEWEB)

Sato-Tomita, Ayana, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Shibayama, Naoya, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Okabe, Takahiro [Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke 329-0498 (Japan); Happo, Naohisa [Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-Ku, Hiroshima 731-3194 (Japan); Kimura, Koji [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Matsushita, Tomohiro [Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Sayo, Hyogo 679-5198 (Japan); Park, Sam-Yong [Drug Design Laboratory, Department of Medical Life Science, Yokohama City University, Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Sasaki, Yuji C. [Department of Advanced Material Science, Graduate School of Frontier Science, The University of Tokyo, Kashiwanoha, Kashiwa 277-8561 (Japan); Hayashi, Kouichi, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan)

2016-06-15

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α{sub 2}β{sub 2} tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm{sup 3}) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

International Nuclear Information System (INIS)

Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard

2007-01-01

Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2 and diffracted to a resolution of 1.6 Å

Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

Energy Technology Data Exchange (ETDEWEB)

Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard, E-mail: irmi.sinning@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF 328, D-69120 Heidelberg (Germany)

2007-09-01

Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 and diffracted to a resolution of 1.6 Å.

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.

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

Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination

International Nuclear Information System (INIS)

Han, Y.; Cang, H.-X.; Zhou, J.-X.; Wang, Y.-P.; Bi, R.-C.; Colelesage, J.; Delbaere, L.T.J.; Nahoum, V.; Shi, R.; Zhou, M.; Zhu, D.-W.; Lin, S.-X.

2004-01-01

The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46 A vs. 1.87 A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2 A 2 vs. 42.9 A 2 ) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing

Crystal structure of the β2 adrenergic receptor-Gs protein complex

DEFF Research Database (Denmark)

Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

2011-01-01

-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

Simulation of diffusion time of small molecules in protein crystals.

Science.gov (United States)

Geremia, Silvano; Campagnolo, Mara; Demitri, Nicola; Johnson, Louise N

2006-03-01

A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.

Protein Crystal Recombinant Human Insulin

Science.gov (United States)

1994-01-01

The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

Crystallization, diffraction data collection and preliminary crystallographic analysis of DING protein from Pseudomonas fluorescens

International Nuclear Information System (INIS)

Moniot, Sebastien; Elias, Mikael; Kim, Donghyo; Scott, Ken; Chabriere, Eric

2007-01-01

Crystallization of DING protein from P. fluorescens is reported. A complete data set was collected to 1.43 Å resolution. PfluDING is a phosphate-binding protein expressed in Pseudomonas fluorescens. This protein is clearly distinct from the bacterial ABC transporter soluble phosphate-binding protein PstS and is more homologous to eukaryotic DING proteins. Interestingly, bacterial DING proteins have only been detected in certain Pseudomonas species. Although DING proteins seem to be ubiquitous in eukaryotes, they are systematically absent from eukaryotic genomic databases and thus are still quite mysterious and poorly characterized. PfluDING displays mitogenic activity towards human cells and binds various ligands such as inorganic phosphate, pyrophosphate, nucleotide triphosphates and cotinine. Here, the crystallization of PfluDING is reported in a monoclinic space group (P2 1 ), with typical unit-cell parameters a = 36.7, b = 123.7, c = 40.8 Å, α = 90, β = 116.7, γ = 90°. Preliminary crystallographic analysis reveals good diffraction quality for these crystals and a 1.43 Å resolution data set has been collected

Liquid nitrogen dewar for protein crystal growth

Science.gov (United States)

2001-01-01

Gaseous Nitrogen Dewar apparatus developed by Dr. Alex McPherson of the University of California, Irvine for use aboard Mir and the International Space Station allows large quantities of protein samples to be crystallized in orbit. The specimens are contained either in plastic tubing (heat-sealed at each end). Biological samples are prepared with a precipitating agent in either a batch or liquid-liquid diffusion configuration. The samples are then flash-frozen in liquid nitrogen before crystallization can start. On orbit, the Dewar is placed in a quiet area of the station and the nitrogen slowly boils off (it is taken up by the environmental control system), allowing the proteins to thaw to begin crystallization. The Dewar is returned to Earth after one to four months on orbit, depending on Shuttle flight opportunities. The tubes then are analyzed for crystal presence and quality

Active Photonic Crystal Waveguides

DEFF Research Database (Denmark)

Ek, Sara

This thesis deals with the fabrication and characterization of active photonic crystal waveguides, realized in III-V semiconductor material with embedded active layers. The platform offering active photonic crystal waveguides has many potential applications. One of these is a compact photonic...... due to photonic crystal dispersion. The observations are explained by the enhancement of net gain by light slow down. Another application based on active photonic crystal waveguides is micro lasers. Measurements on quantum dot micro laser cavities with different mirror configurations and photonic...

Electron crystallography of three dimensional protein crystals

NARCIS (Netherlands)

Georgieva, Dilyana

2008-01-01

This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for

Crystallization and preliminary crystallographic data of the PAS domain of the NifL protein from Azotobacter vinelandii.

NARCIS (Netherlands)

Hefti, M.H.; Hendle, J.; Enroth, C.; Vervoort, J.J.M.; Tucker, P.A.

2001-01-01

The Azotobacter vinelandii NifL protein is a redox-sensing flavoprotein which inhibits the activity of the nitrogen-specific transcriptional activator NifA. The N-terminal PAS domain has been overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method. The crystal

The Growth of Protein Crystals Using McDUCK

Science.gov (United States)

Ewing, Felicia; Wilson, Lori; Nadarajah, Arunan; Pusey, Marc

1998-01-01

Most of the current microgravity crystal growth hardware is optimized to produce crystals within the limited time available on orbit. This often results in the actual nucleation and growth process being rushed or the system not coming to equilibrium within the limited time available. Longer duration hardware exists, but one cannot readily pick out crystals grown early versus those which nucleated and grew more slowly. We have devised a long duration apparatus, the Multi-chamber Dialysis Unit for Crystallization Kinetics, or McDUCK. This apparatus-is a series of protein chambers, stacked upon a precipitant reservoir chamber. All chambers are separated by a dialysis membrane, which serves to pass small molecules while retaining the protein. The volume of the Precipitant chamber is equal to the sum of the volumes of the protein chamber. In operation, the appropriate chambers are filled with precipitant solution or protein solution, and the McDUCK is placed standing upright, with the precipitant chamber on the bottom. The precipitant diffuses upwards over time, with the time to reach equilibration a function of the diffusivity of the precipitant and the overall length of the diffusion pathway. Typical equilibration times are approximately 2-4 months, and one can readily separate rapid from slow nucleation and growth crystals. An advantage on Earth is that the vertical precipitant concentration gradient dominates that of the solute, thus dampening out solute density gradient driven convective flows. However, large Earth-grown crystals have so far tended to be more two dimensional. Preliminary X-ray diffraction analysis of lysozyme crystals grown in McDUCK have indicated that the best, and largest, come from the middle chambers, suggesting that there is an optimal growth rate. Further, the improvements in diffraction resolution have been better signal to noise ratios in the low resolution data, not an increase in resolution overall. Due to the persistently large crystals

Heterogeneous nucleation of protein crystals on fluorinated layered silicate.

Directory of Open Access Journals (Sweden)

Keita Ino

Full Text Available Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna

International Nuclear Information System (INIS)

Kumar, Sanjit; Singh, Nagendra; Sinha, Mau; Kaur, Punit; Srinivasan, A.; Sharma, Sujata; Singh, T. P.

2009-01-01

A novel 15 kDa antifungal protein amaryllin has been crystallized using 30% PEG 8000 as the precipitating agent. The crystals belong to orthorhombic space group I or I2 1 2 1 2 1 with cell dimensions, a = 48.6, b = 61.9 and c = 79.6–Å. A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS–PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress

Approach for growth of high-quality and large protein crystals

Energy Technology Data Exchange (ETDEWEB)

Matsumura, Hiroyoshi, E-mail: matsumura@chem.eng.osaka-u.ac.jp [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Sugiyama, Shigeru; Hirose, Mika; Kakinouchi, Keisuke; Maruyama, Mihoko; Murai, Ryota [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); Adachi, Hiroaki; Takano, Kazufumi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Murakami, Satoshi [JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Mori, Yusuke; Inoue, Tsuyoshi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan)

2011-01-01

Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed. Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein–precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.

Fast high-pressure freezing of protein crystals in their mother liquor

International Nuclear Information System (INIS)

Burkhardt, Anja; Warmer, Martin; Panneerselvam, Saravanan; Wagner, Armin; Zouni, Athina; Glöckner, Carina; Reimer, Rudolph; Hohenberg, Heinrich; Meents, Alke

2012-01-01

Protein crystals were vitrified using high-pressure freezing in their mother liquor at 210 MPa and 77 K without cryoprotectants or oil coating. The method was successfully applied to photosystem II, which is representative of a membrane protein with a large unit cell and weak crystal contacts. High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts

The liquid protein phase in crystallization: a case study—intact immunoglobulins

Science.gov (United States)

Kuznetsov, Yurii G.; Malkin, Alexander J.; McPherson, Alexander

2001-11-01

A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in "salting out" or "salting in' procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The "liquid protein" phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO 4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase.

Crystallization of bi-functional ligand protein complexes.

Science.gov (United States)

Antoni, Claudia; Vera, Laura; Devel, Laurent; Catalani, Maria Pia; Czarny, Bertrand; Cassar-Lajeunesse, Evelyn; Nuti, Elisa; Rossello, Armando; Dive, Vincent; Stura, Enrico Adriano

2013-06-01

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects. Copyright © 2013 Elsevier Inc. All rights reserved.

An Overview of Hardware for Protein Crystallization in a Magnetic Field

Directory of Open Access Journals (Sweden)

Er-Kai Yan

2016-11-01

Full Text Available Protein crystallization under a magnetic field is an interesting research topic because a magnetic field may provide a special environment to acquire improved quality protein crystals. Because high-quality protein crystals are very useful in high-resolution structure determination using diffraction techniques (X-ray, neutron, and electron diffraction, research using magnetic fields in protein crystallization has attracted substantial interest; some studies have been performed in the past two decades. In this research field, the hardware is especially essential for successful studies because the environment is special and the design and utilization of the research apparatus in such an environment requires special considerations related to the magnetic field. This paper reviews the hardware for protein crystallization (including the magnet systems and the apparatus designed for use in a magnetic field and progress in this area. Future prospects in this field will also be discussed.

Can radiation damage to protein crystals be reduced using small-molecule compounds?

Energy Technology Data Exchange (ETDEWEB)

Kmetko, Jan [Kenyon College, Gambier, OH 43022 (United States); Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, NY 14853 (United States); Kenyon College, Gambier, OH 43022 (United States)

2011-10-01

Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

Can radiation damage to protein crystals be reduced using small-molecule compounds?

International Nuclear Information System (INIS)

Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

2011-01-01

Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions

Protein nanocrystallography: growth mechanism and atomic structure of crystals induced by nanotemplates.

Science.gov (United States)

Pechkova, E; Vasile, F; Spera, R; Fiordoro, S; Nicolini, C

2005-11-01

Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant alpha-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation.

The role of shape vs. patches in protein crystallization

Science.gov (United States)

Glaser, Jens; Glotzer, Sharon C.

Proteins fold into a multitude of three-dimensional native structures. The structures of over 100,000 known proteins are deposited in the protein data bank, and most of them have been determined through X-ray crystallography. We ask the question if the role of shape in protein crystallization can be isolated using simulation. Current computational studies show that patchy complementary contacts stabilize experimentally observed P212121 crystal structures for relatively globular protein using spherical protein models. Here we study an anisotropic rigid shape model of green fluorescent protein based on a coarse-grained representation of the atomic coordinates. Using GPU-accelerated molecular dynamics simulations, we find that the experimentally found crystal structure can be stabilized in self-assembly by using complementary attractive patches, confirming the earlier findings. However, we discuss the additional roles of solvent mediated and electrostatic interactions, depletion effects and the self-assembly properties of a purely hard shape model in stabilizing different assemblies. Our findings shed light on fundamental assembly mechanisms in colloidal systems with many competing interactions.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Energy Technology Data Exchange (ETDEWEB)

Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2008-04-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Expression, purification and crystallization of a lyssavirus matrix (M) protein

International Nuclear Information System (INIS)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

Crystallization Process of Protein Rv0731c from Mycobacterium Tuberculosis for a Successful Atomic Resolution Crystal Structure at 1.2 Angstrom

Energy Technology Data Exchange (ETDEWEB)

Zhu, Liang Cong

2009-06-08

Proteins are bio-macromolecules consisting of basic 20 amino acids and have distinct three-dimensional folds. They are essential parts of organisms and participate in every process within cells. Proteins are crucial for human life, and each protein within the body has a specific function, such as antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins and transport proteins. Determining three-dimensional structure of a protein can help researchers discover the remarkable protein folding, binding site, conformation and etc, in order to understand well of protein interaction and aid for possible drug design. The research on protein structure by X-ray protein crystallography carried by Li-Wei Hung's research group in the Physical Bioscience Division at Lawrence Berkeley National Laboratory (LBNL) is focusing on protein crystallography. The research in this lab is in the process of from crystallizing the proteins to determining the three dimensional crystal structures of proteins. Most protein targets are selected from Mycobacterium Tuberculosis. TB (Tuberculosis) is a possible fatal infectious disease. By studying TB target protein can help discover antituberculer drugs, and find treatment for TB. The high-throughput mode of crystallization, crystal harvesting, crystal screening and data collection are applied to the research pipeline (Figure 1). The X-ray diffraction data by protein crystals can be processed and analyzed to result in a three dimensional representation of electron density, producing a detailed model of protein structure. Rv0731c is a conserved hypothetical protein with unknown function from Mycobacterium Tuberculosis. This paper is going to report the crystallization process and brief structure information of Rv0731c.

Two-photon excited UV fluorescence for protein crystal detection

International Nuclear Information System (INIS)

Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

2011-01-01

Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC

Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis

Science.gov (United States)

Leah S. Bauer

1995-01-01

Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the...

Graphene as a protein crystal mounting material to reduce background scatter.

Science.gov (United States)

Wierman, Jennifer L; Alden, Jonathan S; Kim, Chae Un; McEuen, Paul L; Gruner, Sol M

2013-10-01

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

Determination of protein and solvent volumes in protein crystals from contrast variation data

Energy Technology Data Exchange (ETDEWEB)

Badger, J. [Brandeis Univ., Waltham, MA (United States)

1994-12-31

By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited. (1) A translation function that employs contrast variation data gave a sharp minimum within 1-9{Angstrom} of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model. (2) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

Crystal structure of the β₂ adrenergic receptor-Gs protein complex

Energy Technology Data Exchange (ETDEWEB)

Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

2011-12-07

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β₂ adrenergic receptor (β₂AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β₂AR and nucleotide-free Gs heterotrimer. The principal interactions between the β₂AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β₂AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

Science.gov (United States)

Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

1998-01-01

Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

Comparative analysis of amino acids and amino-acid derivatives in protein crystallization

International Nuclear Information System (INIS)

Ito, Len; Shiraki, Kentaro; Yamaguchi, Hiroshi

2010-01-01

New types of aggregation suppressors, such as amino acids and their derivatives, were focused on as fourth-component additives. Data were obtained that indicated that the additives promote protein crystallization. Optimal conditions for protein crystallization are difficult to determine because proteins tend to aggregate in saturated solutions. This study comprehensively evaluates amino acids and amino-acid derivatives as additives for crystallization. This fourth component of the solution increases the probability of crystallization of hen egg-white lysozyme in various precipitants owing to a decrease in aggregation. These results suggest that the addition of certain types of amino acids and amino-acid derivatives, such as Arg, Lys and esterified and amidated amino acids, is a simple method of improving the success rate of protein crystallization

Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

Directory of Open Access Journals (Sweden)

Ellen Wallace

Full Text Available The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive

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

Computer simulations of radiation damage in protein crystals

International Nuclear Information System (INIS)

Zehnder, M.

2007-03-01

The achievable resolution and the quality of the dataset of an intensity data collection for structure analysis of protein crystals with X-rays is limited among other factors by radiation damage. The aim of this work is to obtain a better quantitative understanding of the radiation damage process in proteins. Since radiation damage is unavoidable it was intended to look for the optimum ratio between elastically scattered intensity and radiation damage. Using a Monte Carlo algorithm physical processes after an inelastic photon interaction are studied. The main radiation damage consists of ionizations of the atoms through the electron cascade following any inelastic photon interaction. Results of the method introduced in this investigation and results of an earlier theoretical studies of the influence of Auger-electron transport in diamond are in a good agreement. The dependence of the radiation damage as a function of the energy of the incident photon was studied by computer-aided simulations. The optimum energy range for diffraction experiments on the protein myoglobin is 10-40 keV. Studies of radiation damage as a function of crystal volume and shape revealed that very small plate or rod shaped crystals suffer less damage than crystals formed like a cube with the same volume. Furthermore the influence of a few heavy atoms in the protein molecule on radiation damage was examined. Already two iron atoms in the unit cell of myoglobin increase radiation damage significantly. (orig.)

Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

Science.gov (United States)

Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

2006-09-06

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

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

Expression, purification and crystallization of a lyssavirus matrix (M) protein

Science.gov (United States)

Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

2008-01-01

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state.

Science.gov (United States)

Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

2008-05-01

To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

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.

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state

International Nuclear Information System (INIS)

Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

2008-01-01

Hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0) and the quality of the crystals was characterized. Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25–40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme

Role of Tamm-Horsfall protein and uromodulin in calcium oxalate crystallization

Directory of Open Access Journals (Sweden)

Carvalho M.

2002-01-01

Full Text Available One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys. The aim of the present study was to determine the behavior of two of these proteins, Tamm-Horsfall and uromodulin, in calcium oxalate crystallization in vitro. We studied a group of 10 male stone formers who had formed at least one kidney stone composed of calcium oxalate. They were classified as having idiopathic nephrolithiasis and had no well-known metabolic risk factors involved in kidney stone pathogenesis. Ten normal men were used as controls, as was a group consisting of five normal women and another consisting of five pregnant women. Crystallization was induced by a fixed supersaturation of calcium oxalate and measured with a Coulter Counter. All findings were confirmed by light and scanning electron microscopy. The number of particulate material deposited from patients with Tamm-Horsfall protein was higher than that of the controls (P<0.001. However, Tamm-Horsfall protein decreased the particle diameter of the stone formers when analyzed by the mode of the volume distribution curve (P<0.002 (5.64 ± 0.55 µm compared to 11.41 ± 0.48 µm of uromodulin; 15.94 ± 3.93 µm and 12.45 ± 0.97 µm of normal men Tamm-Horsfall protein and uromodulin, respectively; 8.17 ± 1.57 µm and 9.82 ± 0.95 µm of normal women Tamm-Horsfall protein and uromodulin, respectively; 12.17 ± 1.41 µm and 12.99 ± 0.51 µm of pregnant Tamm-Horsfall protein and uromodulin, respectively. Uromodulin produced fewer particles than Tamm-Horsfall protein in all groups. Nonetheless, the total volume of the crystals produced by uromodulin was higher than that produced by Tamm-Horsfall protein. Our results indicate a different effect of Tamm-Horsfall protein and uromodulin. This dual behavior suggests different functions. Tamm-Horsfall protein may act on nucleation and inhibit crystal aggregation, while

Systematic comparison of crystal and NMR protein structures deposited in the protein data bank.

Science.gov (United States)

Sikic, Kresimir; Tomic, Sanja; Carugo, Oliviero

2010-09-03

Nearly all the macromolecular three-dimensional structures deposited in Protein Data Bank were determined by either crystallographic (X-ray) or Nuclear Magnetic Resonance (NMR) spectroscopic methods. This paper reports a systematic comparison of the crystallographic and NMR results deposited in the files of the Protein Data Bank, in order to find out to which extent these information can be aggregated in bioinformatics. A non-redundant data set containing 109 NMR - X-ray structure pairs of nearly identical proteins was derived from the Protein Data Bank. A series of comparisons were performed by focusing the attention towards both global features and local details. It was observed that: (1) the RMDS values between NMR and crystal structures range from about 1.5 Å to about 2.5 Å; (2) the correlation between conformational deviations and residue type reveals that hydrophobic amino acids are more similar in crystal and NMR structures than hydrophilic amino acids; (3) the correlation between solvent accessibility of the residues and their conformational variability in solid state and in solution is relatively modest (correlation coefficient = 0.462); (4) beta strands on average match better between NMR and crystal structures than helices and loops; (5) conformational differences between loops are independent of crystal packing interactions in the solid state; (6) very seldom, side chains buried in the protein interior are observed to adopt different orientations in the solid state and in solution.

Automating the application of smart materials for protein crystallization.

Science.gov (United States)

Khurshid, Sahir; Govada, Lata; El-Sharif, Hazim F; Reddy, Subrayal M; Chayen, Naomi E

2015-03-01

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as `smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials.

The biochemistry of the protein crystal toxin of Bacillus thuringiensis

Science.gov (United States)

Paul G. Fast

1985-01-01

The crystal consists of dimeric protein subunits. The monomer peptide chains are held together in the subunit and the subunit in the crystal by disulfide and non-covalent bonds. The monomer peptide has a molecular weight of about 130 kdaltons which, in the presence of proteases, is hydrolyzed to a protease-resistant-protein of 65 kda that is toxic both to larvae by...

Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

Science.gov (United States)

Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

1998-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

Protein sensing by nanofluidic crystal and its signal enhancement

Science.gov (United States)

Sang, Jianming; Du, Hongtan; Wang, Wei; Chu, Ming; Wang, Yuedan; Li, Haichao; Alice Zhang, Haixia; Wu, Wengang; Li, Zhihong

2013-01-01

Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel protein sensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing. PMID:24404017

Four crystal forms of a Bence-Jones protein

International Nuclear Information System (INIS)

Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander

2004-01-01

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4 3 2 1 2 and P2 1 2 1 2 1 , with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3 1 21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases

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.

High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

Science.gov (United States)

Kim, Sung-Hou [Moraga, CA; Kim, Rosalind [Moraga, CA; Jancarik, Jamila [Walnut Creek, CA

2012-01-31

An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

International Nuclear Information System (INIS)

Christensen, Jeff; Gerdts, Cory J.; Clifton, Mathew C.; Stewart, Lance

2011-01-01

SSGCID protein crystals were salvaged and stored using the MPCS Plug Maker and CrystalCards when high-throughput traditional sitting-drop vapor diffusion initially failed. The MPCS Plug Maker is a microcapillary-based protein-crystallization system for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long-term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal-salvage and storage method

Protein crystal growth studies at the Center for Macromolecular Crystallography

International Nuclear Information System (INIS)

DeLucas, Lawrence J.; Long, Marianna M.; Moore, Karen M.; Harrington, Michael; McDonald, William T.; Smith, Craig D.; Bray, Terry; Lewis, Johanna; Crysel, William B.; Weise, Lance D.

2000-01-01

The Center for Macromolecular Crystallography (CMC) has been involved in fundamental studies of protein crystal growth (PCG) in microgravity and in our earth-based laboratories. A large group of co-investigators from academia and industry participated in these experiments by providing protein samples and by performing the x-ray crystallographic analysis. These studies have clearly demonstrated the usefulness of a microgravity environment for enhancing the quality and size of protein crystals. Review of the vapor diffusion (VDA) PCG results from nineteen space shuttle missions is given in this paper

In vacuo X-ray data collection from graphene-wrapped protein crystals

Energy Technology Data Exchange (ETDEWEB)

Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Crawshaw, Adam D. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Salgado, Paula S. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

2015-09-26

A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

In vacuo X-ray data collection from graphene-wrapped protein crystals

International Nuclear Information System (INIS)

Warren, Anna J.; Crawshaw, Adam D.; Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana; Salgado, Paula S.; Evans, Gwyndaf

2015-01-01

A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

Science.gov (United States)

Delucas, Lawrence J.; Moore, K. M.; Vanderwoerd, M.; Bray, T. L.; Smith, C.; Carson, M.; Narayana, S. V. L.; Rosenblum, W. M.; Carter, D.; Clark, A. D, Jr.

1994-01-01

Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission.

Four crystal forms of a Bence-Jones protein

Energy Technology Data Exchange (ETDEWEB)

Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander, E-mail: amcphers@uci.edu [Molecular Biology and Biochemistry, University of California, Irvine, 560 Steinhaus Hall, Irvine, CA 92697-3900 (United States)

2005-01-01

Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4{sub 3}2{sub 1}2 and P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3{sub 1}21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

International Nuclear Information System (INIS)

Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

2013-01-01

A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection

Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

Energy Technology Data Exchange (ETDEWEB)

Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi, E-mail: kumasaka@spring8.or.jp [Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

2013-09-01

A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.

Nonclassical nucleation pathways in protein crystallization.

Science.gov (United States)

Zhang, Fajun

2017-11-08

Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

Nonclassical nucleation pathways in protein crystallization

Science.gov (United States)

Zhang, Fajun

2017-11-01

Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

Crystal structure of an activated variant of small heat shock protein Hsp16.5.

Science.gov (United States)

McHaourab, Hassane S; Lin, Yi-Lun; Spiller, Benjamin W

2012-06-26

How does the sequence of a single small heat shock protein (sHSP) assemble into oligomers of different sizes? To gain insight into the underlying structural mechanism, we determined the crystal structure of an engineered variant of Methanocaldococcus jannaschii Hsp16.5 wherein a 14 amino acid peptide from human heat shock protein 27 (Hsp27) was inserted at the junction of the N-terminal region and the α-crystallin domain. In response to this insertion, the oligomer shell expands from 24 to 48 subunits while maintaining octahedral symmetry. Oligomer rearrangement does not alter the fold of the conserved α-crystallin domain nor does it disturb the interface holding the dimeric building block together. Rather, the flexible C-terminal tail of Hsp16.5 changes its orientation relative to the α-crystallin domain which enables alternative packing of dimers. This change in orientation preserves a peptide-in-groove interaction of the C-terminal tail with an adjacent β-sandwich, thereby holding the assembly together. The interior of the expanded oligomer, where substrates presumably bind, retains its predominantly nonpolar character relative to the outside surface. New large windows in the outer shell provide increased access to these substrate-binding regions, thus accounting for the higher affinity of this variant to substrates. Oligomer polydispersity regulates sHSPs chaperone activity in vitro and has been implicated in their physiological roles. The structural mechanism of Hsp16.5 oligomer flexibility revealed here, which is likely to be highly conserved across the sHSP superfamily, explains the relationship between oligomer expansion observed in disease-linked mutants and changes in chaperone activity.

Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

Science.gov (United States)

Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

2013-01-01

Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. PMID:23999307

Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

Science.gov (United States)

Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

2010-01-01

In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

International Nuclear Information System (INIS)

Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

2015-01-01

An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s −1 ) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening

Protein crystals as scanned probes for recognition atomic force microscopy.

Science.gov (United States)

Wickremasinghe, Nissanka S; Hafner, Jason H

2005-12-01

Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

Energy Technology Data Exchange (ETDEWEB)

Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

2015-01-01

An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Science.gov (United States)

Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1

International Nuclear Information System (INIS)

Meulenbroek, Elisabeth M.; Pannu, Navraj S.

2011-01-01

Human Cockayne syndrome protein A has been cocrystallized with human DNA damage-binding protein 1 and data have been collected to 2.9 Å resolution. Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility

Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

DEFF Research Database (Denmark)

Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

2006-01-01

A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

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

Expression, crystallization and preliminary crystallographic analysis of C-reactive protein from zebrafish

International Nuclear Information System (INIS)

Chen, Rong; Qi, Jianxun; Yao, Shugang; Pan, Xiaocheng; Gao, Feng; Xia, Chun

2011-01-01

Crystals of native and selenomethionine-substituted C-reactive protein from zebrafish diffracted to 2.3 and 1.7 Å resolution, respectively, and belonged to space group R3 with one molecule per asymmetric unit. The Matthews coefficient was calculated to be 3.28 Å 3 Da −1 . C-reactive protein (CRP) is an acute phase protein that is found in blood, the concentration of which in plasma rises rapidly in response to inflammation. It functions as a pattern-recognition molecule, recognizing dead cells and various pathogenic agents and eliminating them by utilizing the classical complement pathway and activating macrophages. CRP is phylogenetically highly conserved in invertebrates and mammals. To date, information on the CRP gene has been reported from numerous species of animals, but little is known about the structure of CRP from species other than humans. In order to solve the structure of CRP from bony fish, the CRP gene from zebrafiah (Danio rerio) was cloned and expressed in Escherichia coli. The zebrafish CRP (Dare-CRP) was then purified and crystallized. The crystal diffracted to 2.3 Å resolution and belonged to space group R3, with unit-cell parameters a = b = 114.7, c = 61.0 Å. The Matthews coefficient and solvent content were calculated to be 3.28 Å 3 Da −1 and 62.55%, respectively. Determination of the zebrafish CRP structure should be helpful in investigating the evolution of CRPs in the innate immune system

Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents.

Directory of Open Access Journals (Sweden)

Anil S Thakur

2007-10-01

Full Text Available Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed.We tested the effect of nine potential heterogenous nucleating agents on crystallization of ten test proteins in a sparse matrix screen. Several nucleating agents induced crystal formation under conditions where no crystallization occurred in the absence of the nucleating agent. Four nucleating agents: dried seaweed; horse hair; cellulose and hydroxyapatite, had a considerable overall positive effect on crystallization success. This effect was further enhanced when these nucleating agents were used in combination with each other.Our results suggest that the addition of heterogeneous nucleating agents increases the chances of crystal formation when using sparse matrix screens.

Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

Science.gov (United States)

Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

2012-05-01

Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

International Nuclear Information System (INIS)

Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

2005-01-01

Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Crystal Structure of a Lipid G Protein-Coupled Receptor

Energy Technology Data Exchange (ETDEWEB)

Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C [Scripps; (Receptos)

2012-03-01

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P₁-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P₁, resulting in the modulation of immune and stromal cell responses.

A chemical library to screen protein and protein-ligand crystallization using a versatile microfluidic platform

OpenAIRE

Gerard , Charline ,; Ferry , Gilles; Vuillard , Laurent ,; Boutin , Jean ,; Ferte , Nathalie ,; Grossier , Romain ,; Candoni , Nadine ,; Veesler , Stéphane ,

2018-01-01

Here, we describe a plug-and-play microfluidic platform, suitable for protein crystallization. The droplet factory is designed to generate hundreds of droplets as small as a few nanoliters (2 to 10nL) for screening and optimization of crystallization conditions. Commercially-available microfluidic junctions and tubing are combined to create the appropriate geometry. In addition, a " chemical library " is produced in tubing. The microfluidic geometry for a " crystallization agent-based chemica...

Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

Science.gov (United States)

Ramya, L; Ramakrishnan, Vigneshwar

2016-07-01

Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins.

Directory of Open Access Journals (Sweden)

Jan Kubicek

Full Text Available We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i the stabilization of membrane proteins in the meso phase, (ii the control of hydration level and additive concentration by vapor diffusion. The new technology (iii significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII from Halobacterium salinarum for the first time.

Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris

International Nuclear Information System (INIS)

Schultz-Heienbrok, Robert; Remmel, Natascha; Klingenstein, R.; Rossocha, Maksim; Sandhoff, Konrad; Saenger, Wolfram; Maier, Timm

2006-01-01

Three different crystal forms were obtained of human saposin C. The structures could not be determined by molecular replacement using known solution structures of the protein as search models, supporting the notion of a highly flexible protein. The amphiphilic saposin proteins (A, B, C and D) act at the lipid–water interface in lysosomes, mediating the hydrolysis of membrane building blocks by water-soluble exohydrolases. Human saposin C activates glucocerebrosidase and β-galactosylceramidase. The protein has been expressed in Pichia pastoris, purified and crystallized in three different crystal forms, diffracting to a maximum resolution of 2.5 Å. Hexagonal crystals grew from 2-propanol-containing solution and contain a single molecule in the asymmetric unit according to the Matthews coefficient. Orthorhombic and tetragonal crystals were both obtained with pentaerythritol ethoxylate and are predicted to contain two molecules in the asymmetric unit. Attempts to determine the respective crystal structures by molecular replacement using either the known NMR structure of human saposin C or a related crystal structure as search models have so far failed. The failure of the molecular-replacement method is attributed to conformational changes of the protein, which are known to be required for its biological activity. Crystal structures of human saposin C therefore might be the key to mapping out the conformational trajectory of saposin-like proteins

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

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

A hydrazone Schiff base single crystal (E)-Methyl N"′-(3,4,5-trimethoxybenzylidene) hydrazine carboxylate: Physicochemical, in vitro investigation of antimicrobial activities and molecular docking with DNA gyrase protein

International Nuclear Information System (INIS)

Gomathi, G.; Gopalakrishnan, R.

2016-01-01

Hydrazone Schiff bases have been widely explored for their antimicrobial, anticancer, anticonvulsant properties. The aim of the present work is to investigate the spectroscopic, electrochemical, thermal properties, in vitro study of antimicrobial activity and molecular docking studies of the MBHC compound. Slow evaporation solution growth technique was used to grow the single crystal of the MBHC compound. Single crystal X-ray diffraction, FTIR and FT-Raman spectroscopic studies are performed and confirmed the grown MBHC compound. UV–Vis spectroscopy and electrochemical studies deduced the absorption region and HOMO-LUMO band gap value of the compound. Resazurin reduction assay method was utilized to perform antibacterial and antifungal studies which resulted in lesser effectiveness of the MBHC compound compared to the erythromycin and fluconazole tablets. Molecular docking of the MBHC compound with the DNA gyrase protein exhibited the good binding affinity with energy of − 43.196 kcal/mol and docking score of − 6.266 and having good interaction with aminoacids – ASP81 and ARG84. - Highlights: • MBHC single crystal was grown by employing slow evaporation solution growth technique. • The compound crystallizes in monoclinic crystal system with space group P2_1/c. • The HOMO-LUMO band gap value was found to be 1.96 eV. • The compound has lesser antimicrobial activity when compared to erythromycin and fluconazole. • MBHC shows better binding affinity towards DNA gyrase protein.

Protein crystal structure analysis using synchrotron radiation at atomic resolution

International Nuclear Information System (INIS)

Nonaka, Takamasa

1999-01-01

We can now obtain a detailed picture of protein, allowing the identification of individual atoms, by interpreting the diffraction of X-rays from a protein crystal at atomic resolution, 1.2 A or better. As of this writing, about 45 unique protein structures beyond 1.2 A resolution have been deposited in the Protein Data Bank. This review provides a simplified overview of how protein crystallographers use such diffraction data to solve, refine, and validate protein structures. (author)

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

Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

Energy Technology Data Exchange (ETDEWEB)

Chen, Hui; Mustafi, Sourajit M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); LeMaster, David M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States); Li, Zhong [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); Héroux, Annie [Brookhaven National Laboratory, Upton, NY 11973 (United States); Li, Hongmin; Hernández, Griselda, E-mail: griselda@wadsworth.org [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States)

2014-03-01

Two crystal forms of unligated FKBP12.6 exhibit multiple conformations in the active site and in the 80s loop, the primary site for known protein-recognition interactions. The previously unreported NMR backbone assignment of FKBP12.6 revealed extensive doubling of amide resonances, which reflects a slow conformational transition centered in the 80s loop. The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca{sup 2+} channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2{sub 1} and P3{sub 1}21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3{sub 1}21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition ‘80s loop’ is flipped in the P2{sub 1} crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

Science.gov (United States)

Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

2014-11-01

X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

KAUST Repository

Dumbrack, Roland

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

Two types of amorphous protein particles facilitate crystal nucleation.

Science.gov (United States)

Yamazaki, Tomoya; Kimura, Yuki; Vekilov, Peter G; Furukawa, Erika; Shirai, Manabu; Matsumoto, Hiroaki; Van Driessche, Alexander E S; Tsukamoto, Katsuo

2017-02-28

Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

Understanding water equilibration fundamentals as a step for rational protein crystallization.

Directory of Open Access Journals (Sweden)

Pedro M Martins

Full Text Available BACKGROUND: Vapor diffusion is the most widely used technique for protein crystallization and the rate of water evaporation plays a key role on the quality of the crystals. Attempts have been made in the past to solve the mass transfer problem governing the evaporation process, either analytically or by employing numerical methods. Despite these efforts, the methods used for protein crystallization remain based on trial and error techniques rather than on fundamental principles. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a new theoretical model which describes the hanging drop method as a function of the different variables that are known to influence the evaporation process. The model is extensively tested against experimental data published by other authors and considering different crystallizing conditions. Aspects responsible for the discrepancies between the existing theories and the measured evaporation kinetics are especially discussed; they include the characterization of vapor-liquid equilibrium, the role of mass transfer within the evaporating droplet, and the influence of the droplet-reservoir distance. CONCLUSIONS/SIGNIFICANCE: The validation tests show that the proposed model can be used to predict the water evaporation rates under a wide range of experimental conditions used in the hanging drop vapor-diffusion method, with no parameter fitting or computational requirements. This model combined with protein solubility data is expected to become a useful tool for a priori screening of crystallization conditions.

Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

Energy Technology Data Exchange (ETDEWEB)

Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

1994-12-31

The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

International Nuclear Information System (INIS)

Timmins, P.A.; Pebay-Peyroula, E.

1994-01-01

The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H 2 O/D 2 O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished

Crystal structure of Helicobacter pylori neutrophil-activating protein with a di-nuclear ferroxidase center in a zinc or cadmium-bound form

Energy Technology Data Exchange (ETDEWEB)

Yokoyama, Hideshi, E-mail: h-yokoya@u-shizuoka-ken.ac.jp [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Tsuruta, Osamu; Akao, Naoya; Fujii, Satoshi [School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

2012-06-15

Highlights: Black-Right-Pointing-Pointer Structures of a metal-bound Helicobacter pylori neutrophil-activating protein were determined. Black-Right-Pointing-Pointer Two zinc ions were tetrahedrally coordinated by ferroxidase center (FOC) residues. Black-Right-Pointing-Pointer Two cadmium ions were coordinated in a trigonal-bipyramidal and octahedral manner. Black-Right-Pointing-Pointer The second metal ion was more weakly coordinated than the first at the FOC. Black-Right-Pointing-Pointer A zinc ion was found in one negatively-charged pore suitable as an ion path. -- Abstract: Helicobacter pylori neutrophil-activating protein (HP-NAP) is a Dps-like iron storage protein forming a dodecameric shell, and promotes adhesion of neutrophils to endothelial cells. The crystal structure of HP-NAP in a Zn{sup 2+}- or Cd{sup 2+}-bound form reveals the binding of two zinc or two cadmium ions and their bridged water molecule at the ferroxidase center (FOC). The two zinc ions are coordinated in a tetrahedral manner to the conserved residues among HP-NAP and Dps proteins. The two cadmium ions are coordinated in a trigonal-bipyramidal and distorted octahedral manner. In both structures, the second ion is more weakly coordinated than the first. Another zinc ion is found inside of the negatively-charged threefold-related pore, which is suitable for metal ions to pass through.

Solution structure and dynamics of melanoma inhibitory activity protein

International Nuclear Information System (INIS)

Lougheed, Julie C.; Domaille, Peter J.; Handel, Tracy M.

2002-01-01

Melanoma inhibitory activity (MIA) is a small secreted protein that is implicated in cartilage cell maintenance and melanoma metastasis. It is representative of a recently discovered family of proteins that contain a Src Homologous 3 (SH3) subdomain. While SH3 domains are normally found in intracellular proteins and mediate protein-protein interactions via recognition of polyproline helices, MIA is single-domain extracellular protein, and it probably binds to a different class of ligands.Here we report the assignments, solution structure, and dynamics of human MIA determined by heteronuclear NMR methods. The structures were calculated in a semi-automated manner without manual assignment of NOE crosspeaks, and have a backbone rmsd of 0.38 A over the ordered regions of the protein. The structure consists of an SH3-like subdomain with N- and C-terminal extensions of approximately 20 amino acids each that together form a novel fold. The rmsd between the solution structure and our recently reported crystal structure is 0.86 A over the ordered regions of the backbone, and the main differences are localized to the most dynamic regions of the protein. The similarity between the NMR and crystal structures supports the use of automated NOE assignments and ambiguous restraints to accelerate the calculation of NMR structures

Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

Science.gov (United States)

Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

2011-01-01

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5–12 new techniques learned, which are transferrable to graduate research in academia and industry. PMID:21691428

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°

The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.

Science.gov (United States)

Iacovache, Ioan; Biasini, Marco; Kowal, Julia; Kukulski, Wanda; Chami, Mohamed; van der Goot, F Gisou; Engel, Andreas; Rémigy, Hervé-W

2010-03-01

Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution. (c) 2009 Elsevier Inc. All rights reserved.

Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

International Nuclear Information System (INIS)

Paterson, Neil G.; Riboldi-Tunicliffe, Alan; Mitchell, Timothy J.; Isaacs, Neil W.

2006-01-01

The choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V M = 2.77 Å 3 Da −1 ) and shows a diffraction limit of 3.5 Å

Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

Energy Technology Data Exchange (ETDEWEB)

Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunicliffe, 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 choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V{sub M} = 2.77 Å{sup 3} Da{sup −1}) and shows a diffraction limit of 3.5 Å.

TargetCrys: protein crystallization prediction by fusing multi-view features with two-layered SVM.

Science.gov (United States)

Hu, Jun; Han, Ke; Li, Yang; Yang, Jing-Yu; Shen, Hong-Bin; Yu, Dong-Jun

2016-11-01

The accurate prediction of whether a protein will crystallize plays a crucial role in improving the success rate of protein crystallization projects. A common critical problem in the development of machine-learning-based protein crystallization predictors is how to effectively utilize protein features extracted from different views. In this study, we aimed to improve the efficiency of fusing multi-view protein features by proposing a new two-layered SVM (2L-SVM) which switches the feature-level fusion problem to a decision-level fusion problem: the SVMs in the 1st layer of the 2L-SVM are trained on each of the multi-view feature sets; then, the outputs of the 1st layer SVMs, which are the "intermediate" decisions made based on the respective feature sets, are further ensembled by a 2nd layer SVM. Based on the proposed 2L-SVM, we implemented a sequence-based protein crystallization predictor called TargetCrys. Experimental results on several benchmark datasets demonstrated the efficacy of the proposed 2L-SVM for fusing multi-view features. We also compared TargetCrys with existing sequence-based protein crystallization predictors and demonstrated that the proposed TargetCrys outperformed most of the existing predictors and is competitive with the state-of-the-art predictors. The TargetCrys webserver and datasets used in this study are freely available for academic use at: http://csbio.njust.edu.cn/bioinf/TargetCrys .

Escherichia coli PII protein: purification, crystallization and oligomeric structure.

Science.gov (United States)

Vasudevan, S G; Gedye, C; Dixon, N E; Cheah, E; Carr, P D; Suffolk, P M; Jeffrey, P D; Ollis, D L

1994-01-17

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

Science.gov (United States)

Birdsall, Robert E.; Koshel, Brooke M.; Hua, Yimin; Ratnayaka, Saliya N.; Wirth, Mary J.

2013-01-01

Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization. PMID:23229163

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°

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°.

Implication of Crystal Water Molecules in Inhibitor Binding at ALR2 Active Site

Directory of Open Access Journals (Sweden)

Hymavati

2012-01-01

Full Text Available Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the efficiency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding.

A hydrazone Schiff base single crystal (E)-Methyl N{sup ′}-(3,4,5-trimethoxybenzylidene) hydrazine carboxylate: Physicochemical, in vitro investigation of antimicrobial activities and molecular docking with DNA gyrase protein

Energy Technology Data Exchange (ETDEWEB)

Gomathi, G.; Gopalakrishnan, R., E-mail: krgkrishnan@annauniv.edu

2016-07-01

Hydrazone Schiff bases have been widely explored for their antimicrobial, anticancer, anticonvulsant properties. The aim of the present work is to investigate the spectroscopic, electrochemical, thermal properties, in vitro study of antimicrobial activity and molecular docking studies of the MBHC compound. Slow evaporation solution growth technique was used to grow the single crystal of the MBHC compound. Single crystal X-ray diffraction, FTIR and FT-Raman spectroscopic studies are performed and confirmed the grown MBHC compound. UV–Vis spectroscopy and electrochemical studies deduced the absorption region and HOMO-LUMO band gap value of the compound. Resazurin reduction assay method was utilized to perform antibacterial and antifungal studies which resulted in lesser effectiveness of the MBHC compound compared to the erythromycin and fluconazole tablets. Molecular docking of the MBHC compound with the DNA gyrase protein exhibited the good binding affinity with energy of − 43.196 kcal/mol and docking score of − 6.266 and having good interaction with aminoacids – ASP81 and ARG84. - Highlights: • MBHC single crystal was grown by employing slow evaporation solution growth technique. • The compound crystallizes in monoclinic crystal system with space group P2{sub 1}/c. • The HOMO-LUMO band gap value was found to be 1.96 eV. • The compound has lesser antimicrobial activity when compared to erythromycin and fluconazole. • MBHC shows better binding affinity towards DNA gyrase protein.

Enhancement of nucleation during hanging drop protein crystallization using HF treatment of cover glasses

Energy Technology Data Exchange (ETDEWEB)

Guo, Yun-Zhu; Yin, Da-Chuan; Lu, Qin-Qin; Wang, Xi-Kai; Liu, Jun [Key Laboratory for Space Bioscience and Biotechnology, Faculty of Life Sciences, Northwestern Polytechnical University, Xi' an 710072, Shaanxi (China)

2010-02-15

We examined a simple approach, i.e., etching cover glasses using hydrofluoric acid (HF), to determine whether cover glass treatment enhances nucleation in hanging drop protein crystallization. Hen egg white lysozyme and proteinase K were used as the model proteins. We found that the treatment increased the success rate of crystallization. The results indicated that the simple treatment, which is easy to adopt without changing much in the hanging drop method, can be utilized as an alternative method to enhance protein crystallization screens (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum)

International Nuclear Information System (INIS)

Sharma, Urvashi; Suresh, C. G.

2011-01-01

The purification, characterization and crystallization of a trypsin inhibitor protein isolated from chickpea seeds are reported. A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS–PAGE. The IC 50 value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2 1 2 1 2 form A, P2 1 2 1 2 form B and P2 1 2 1 2 1 . The crystals of P2 1 2 1 2 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å 3 Da −1 , corresponding to a solvent content of 42%. The other two crystal forms (P2 1 2 1 2 form B and P2 1 2 1 2 1 ) diffracted comparatively poorly

The Kinetics of Crystallization of Colloids and Proteins: A Light Scattering Study

Science.gov (United States)

McClymer, Jim

2002-01-01

Hard-sphere colloidal systems serve as model systems for aggregation, nucleation, crystallization and gelation as well as interesting systems in their own right.There is strong current interest in using colloidal systems to form photonic crystals. A major scientific thrust of NASA's microgravity research is the crystallization of proteins for structural determination. The crystallization of proteins is a complicated process that requires a great deal of trial and error experimentation. In spite of a great deal of work, "better" protein crystals cannot always be grown in microgravity and conditions for crystallization are not well understood. Crystallization of colloidal systems interacting as hard spheres and with an attractive potential induced by entropic forces have been studied in a series of static light scattering experiments. Additionally, aggregation of a protein as a function of pH has been studied using dynamic light scattering. For our experiments we used PMMA (polymethylacrylate) spherical particles interacting as hard spheres, with no attractive potential. These particles have a radius of 304 nanometers, a density of 1.22 gm/ml and an index of refraction of 1.52. A PMMA colloidal sample at a volume fraction of approximately 54% was index matched in a solution of cycloheptyl bromide (CHB) and cis-decalin. The sample is in a glass cylindrical vial that is placed in an ALV static and dynamic light scattering goniometer system. The vial is immersed in a toluene bath for index matching to minimize flair. Vigorous shaking melts any colloidal crystals initially present. The sample is illuminated with diverging laser light (632.8 nanometers) from a 4x microscope objective placed so that the beam is approximately 1 cm in diameter at the sample location. The sample is rotated about its long axis at approximately 3.5 revolutions per minute (highest speed) as the colloidal crystal system is non-ergodic. The scattered light is detected at various angles using the

Crystallization and preliminary crystallographic analysis of merohedrally twinned crystals of MJ0729, a CBS-domain protein from Methanococcus jannaschii

International Nuclear Information System (INIS)

Fernández-Millán, Pablo; Kortazar, Danel; Lucas, María; Martínez-Chantar, María Luz; Astigarraga, Egoitz; Fernández, José Andrés; Sabas, Olatz; Albert, Armando; Mato, Jose M.; Martínez-Cruz, Luis Alfonso

2008-01-01

Trigonal crystals of MJ0729 showing different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning were obtained upon slight variation of the pH. CBS domains are small protein motifs, usually associated in tandem, that are implicated in binding to adenosyl groups. Several genetic diseases in humans have been associated with mutations in CBS sequences, which has made them very promising targets for rational drug design. Trigonal crystals of the CBS-domain protein MJ0729 from Methanococcus jannaschii were grown by the vapour-diffusion method at acidic pH. Preliminary analysis of nine X-ray diffraction data sets using Yeates statistics and Britton plots showed that slight variation in the pH as well as in the buffer used in the crystallization experiments led to crystals with different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning

Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein.

Science.gov (United States)

Tanaka, Yoshiki; Iwaki, Shigehiro; Tsukazaki, Tomoya

2017-09-05

The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystallization and preliminary crystallographic characterization of the extrinsic PsbP protein of photosystem II from Spinacia oleracea

International Nuclear Information System (INIS)

Kohoutová, J.; Kutá Smatanová, I.; Brynda, J.; Lapkouski, M.; Revuelta, J. L.; Arellano, J. B.; Ettrich, R.

2009-01-01

Degradation-free crystalization of thrombin-digested recombinant His-tagged PsbP protein of photosystem II from Spinacia oleracea resulting in crystals diffracting to 2.06 Å. Preliminary X-ray diffraction analysis of the extrinsic PsbP protein of photosystem II from spinach (Spinacia oleracea) was performed using N-terminally His-tagged recombinant PsbP protein overexpressed in Escherichia coli. Recombinant PsbP protein (thrombin-digested recombinant His-tagged PsbP) stored in bis-Tris buffer pH 6.00 was crystallized using the sitting-drop vapour-diffusion technique with PEG 550 MME as a precipitant and zinc sulfate as an additive. SDS–PAGE analysis of a dissolved crystal showed that the crystals did not contain the degradation products of recombinant PsbP protein. PsbP crystals diffracted to 2.06 Å resolution in space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.68, b = 46.73, c = 88.9 Å

Models of protein-ligand crystal structures: trust, but verify.

Science.gov (United States)

Deller, Marc C; Rupp, Bernhard

2015-09-01

X-ray crystallography provides the most accurate models of protein-ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein-ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein-ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein-ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein-ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein-ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein-ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

International Nuclear Information System (INIS)

Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

2008-01-01

The biotin protein ligase from S. aureus has been overexpressed in E. coli, purified, crystallized by the hanging-drop vapour-diffusion method and analysed using X-ray diffraction. Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4 2 2 1 2, with unit-cell parameters a = b = 93.665, c = 131.95

A convenient tool for gas derivatization using fine-needle capillary mounting for protein crystals

International Nuclear Information System (INIS)

Mizuno, Nobuhiro; Makino, Masatomo; Kumasaka, Takashi

2013-01-01

A convenient gas-derivatization tool for protein crystals is presented in combination with a fine-needle capillary and a gas-pressure regulator. Gas derivatization of protein crystals is useful not only to analyse gas-binding proteins but also to solve the phase problem of X-ray crystallography by using noble gases. However, the gas pressurization tools for these experiments are often elaborate and need to release the gas before flash-cooling. To simplify this step, a procedure using a fine-needle capillary to mount and flash-cool protein crystals under the pressurization of gases has been developed. After the crystals are picked up with the capillary, the capillary is sealed with an adhesive and then connected directly to a gas regulator. The quality of the diffraction data using this method is comparable with that of data from conventional pressurization procedures. The preparation of xenon-derivatives of hen egg-white lysozyme using this method was a success. In the derivatives, two new xenon binding sites were found and one of their sites vanished by releasing the gas. This observation shows the availability of flash-cooling under gas pressurization. This procedure is simple and useful for preparing gas-derivative crystals

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

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs.

Science.gov (United States)

Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

2014-06-01

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus

DEFF Research Database (Denmark)

Wilkens, Casper; Poulsen, Jens-Christian Navarro; Ramløv, Hans

2014-01-01

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform......, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18...... equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge...

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°

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

International Nuclear Information System (INIS)

Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

2013-01-01

A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

Energy Technology Data Exchange (ETDEWEB)

Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Armour, Wes [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Oxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG (United Kingdom); Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Horrell, Sam [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); University of Liverpool, Liverpool L69 3BX (United Kingdom); McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

2013-07-01

A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

Characterization of the Protein Crystal Growth Apparatus for Microgravity Aboard the Space Station

Science.gov (United States)

Kundrot, Craig E.; Roeber, D.; Achari, A.; Stinson, Thomas N. (Technical Monitor)

2002-01-01

We have conducted experiments to determine the equilibration rates of some major precipitants used in protein crystallography aboard the International Space Station (ISS). The solutions were placed in the Protein Crystallization Apparatus for Microgravity (PCAM) which mimic Cryschem sitting drop trays. The trays were placed in cylinders. These cylinders were placed inside a Single locker Thermal Enclosure System (STES), and were activated for different durations during the flight. Bumpers pressed against elastomers seal drops in a deactivated state during pre-flight and prior to transfer to the ISS. Activation occurs while in flight on the ISS by releasing the bumpers allowing the drops to be exposed to the reservoir. PCAM was flown to the ISS on STS 100, Flight 6A, on April 19, 2001. Six series of equilibration experiments were tested for each precipitant with a small amount of Green Fluorescent Protein (GFP). Cylinder 10 was never activated, 7 was activated for 40 days, 8 was activated for 20 days, 9 was activated for 10 days, 11 was activated for 4 days and 12 was activated for 2 days. Upon the return to Earth by STS 104 on July 24,2001 the samples were transferred to Marshall Space Flight Center. The samples were then brought to the lab and the volumes of each sample were measured.

Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

Energy Technology Data Exchange (ETDEWEB)

Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, George Fu, E-mail: gaof@im.ac.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

2008-08-01

Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

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.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

KAUST Repository

Kim, Jeong Joo

2016-04-09

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation

KAUST Repository

Kim, Jeong  Joo; Lorenz, Robin; Arold, Stefan T.; Reger, Albert  S.; Sankaran, Banumathi; Casteel, Darren  E.; Herberg, Friedrich  W.; Kim, Choel

2016-01-01

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG. Kim et al. obtain the first crystal structure of the PKG I R domain bound with cGMP representing its activated state. It reveals a symmetric R dimer where cGMP molecules provide dimeric contacts. This R-R interaction prevents the high-affinity inhibitory interaction between R-C domain and sustains activation. © 2016 Elsevier Ltd.

Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

International Nuclear Information System (INIS)

Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

2007-01-01

A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

Modification of calcite crystal growth by abalone shell proteins: an atomic force microscope study.

OpenAIRE

Walters, D A; Smith, B L; Belcher, A M; Paloczi, G T; Stucky, G D; Morse, D E; Hansma, P K

1997-01-01

A family of soluble proteins from the shell of Haliotis rufescens was introduced over a growing calcite crystal being scanned in situ by an atomic force microscope (AFM). Atomic step edges on the crystal surface were altered in shape and speed of growth by the proteins. Proteins attached nonuniformly to the surface, indicating different interactions with crystallographically different step edges. The observed changes were consistent with the habit modification induced by this family of protei...

Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

International Nuclear Information System (INIS)

Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

2013-01-01

Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

Cloning and characterization of an insecticidal crystal protein gene ...

Indian Academy of Sciences (India)

Unknown

The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus .... diet or by topical application on food substrates as .... has very high similarity (99.74%) at DNA level with.

Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

International Nuclear Information System (INIS)

Tong, Xiaohang; Ma, Yanlin; Li, Xuemei

2010-01-01

The C-terminal domain of mouse hepatitis virus nucleocapsid protein has been overexpressed in E. coli, purified and crystallized. The crystal belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å, and diffracted to 2.20 Å resolution. Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (V M = 2.16 Å 3 Da −1 )

Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

Directory of Open Access Journals (Sweden)

Marija Luić

2016-06-01

Full Text Available It is generaly believed that enzymes retain most of their functionality in the crystal form due to the large solvent content of protein crystals. This is facilitated by the fact that their natural environment in solution is not too far from the one found in the crystal form. Nevertheless, if the nature of the enzyme is such to require conformational changes, overcoming of the crystal packing constraints may prove to be too difficult. Such conformational change is present in one class of enzymes (purine nucleoside phosphorylases, that is the subject of our scientific interest for many years. The influence of crystal symmetry and crystal packing on the conformation of the active sites in the case of homohexameric purine nucleoside phosphorylases is presented and analysed. This work is licensed under a Creative Commons Attribution 4.0 International License.

Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)

International Nuclear Information System (INIS)

Marr, Eric; Tardie, Mark; Carty, Maynard; Brown Phillips, Tracy; Wang, Ing-Kae; Soeller, Walt; Qiu, Xiayang; Karam, George

2006-01-01

The crystal structure of human adipocyte lipid-binding protein (aP2) with a bound palmitate is reported at 1.5 Å resolution. Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity

Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans

International Nuclear Information System (INIS)

Feng, Ming-Jing; Fu, Tian-Min; Liu, Xiang; Li, Lan-Fen

2010-01-01

SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å. Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°

Effect of ultraviolet and gamma rays on the activity of delta-endotoxin protein crystals of Bacillus thuringiensis

International Nuclear Information System (INIS)

Burges, H.D.; Hillyer, S.; Chanter, D.O.

1975-01-01

Sensitive bioassays with larvae of Pieris brassicae revealed no reduction of insecticidal activity as a result of severe gamma or ultraviolet irradiation of crystals of Bacillus thuringiensis (serotype V). The measured response was the inhibition of larval feeding by the crystals over exposure periods short enough for the presence of live spores not to influence feeding. The results were analyzed using a logistic model. (U.S.)

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%

ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

Science.gov (United States)

Hungler, Arnaud; Momin, Afaque; Diederichs, Kay; Arold, Stefan, T.

2016-01-01

Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for ‘crystallization and preliminary X-ray analysis’ publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. PMID:27980519

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

Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

Directory of Open Access Journals (Sweden)

Xiuli Lu

2013-02-01

Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

Microseed matrix screening for optimization in protein crystallization: what have we learned?

Science.gov (United States)

D'Arcy, Allan; Bergfors, Terese; Cowan-Jacob, Sandra W; Marsh, May

2014-09-01

Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

Use of Plastic Capillaries for Macromolecular Crystallization

Science.gov (United States)

Potter, Rachel R.; Hong, Young-Soo; Ciszak, Ewa M.

2003-01-01

Methods of crystallization of biomolecules in plastic capillaries (Nalgene 870 PFA tubing) are presented. These crystallization methods used batch, free-interface liquid- liquid diffusion alone, or a combination with vapor diffusion. Results demonstrated growth of crystals of test proteins such as thaumatin and glucose isomerase, as well as protein studied in our laboratory such dihydrolipoamide dehydrogenase. Once the solutions were loaded in capillaries, they were stored in the tubes in frozen state at cryogenic temperatures until the desired time of activation of crystallization experiments.

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

International Nuclear Information System (INIS)

Liu, Ting; Shuai, Yingli; Zhou, Honggang

2011-01-01

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

In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

Science.gov (United States)

Moreland, K. Trent; Hong, Mina; Lu, Wenfu; Rowley, Christopher W.; Ornitz, David M.; De Yoreo, James J.; Thalmann, Ruediger

2014-01-01

Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia. PMID:24748133

Cloning, crystallization and preliminary X-ray studies of XC2981 from Xanthomonas campestris, a putative CutA1 protein involved in copper-ion homeostasis

International Nuclear Information System (INIS)

Lin, Chien-Hung; Chin, Ko-Hsin; Gao, Fei Philip; Lyu, Ping-Chiang; Shr, Hui-Lin; Wang, Andrew H.-J.; Chou, Shan-Ho

2006-01-01

A probable copper-ion tolerance protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. Divalent metal ions play key roles in all living organisms, serving as cofactors for many proteins involved in a variety of electron-transfer activities. However, copper ions are highly toxic when an excessive amount is accumulated in a cell. CutA1 is a protein found in all kingdoms of life that is believed to participate in copper-ion tolerance in Escherichia coli, although its specific function remains unknown. Several crystal structures of multimeric CutA1 with different rotation angles and degrees of interaction between trimer interfaces have been reported. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC2981, a possible CutA1 protein present in the plant pathogen Xanthomonas campestris, are reported. The XC2981 crystals diffracted to a resolution of 2.6 Å. They are cubic and belong to space group I23, with unit-cell parameters a = b = c = 130.73 Å

Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation

International Nuclear Information System (INIS)

Gai, Zuoqi; Nakamura, Akiyoshi; Tanaka, Yoshikazu; Hirano, Nagisa; Tanaka, Isao; Yao, Min

2013-01-01

Crystals of a member of the DING protein family (HPBP) were obtained accidentally, and the structure was determined at 1.35 Å resolution. For further analysis, a system for preparation of HPBP was constructed and the structure of a prepared sample was confirmed by X-ray crystal structure analysis at 1.03 Å resolution. After crystallization of a certain protein–RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly

Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation

Energy Technology Data Exchange (ETDEWEB)

Gai, Zuoqi; Nakamura, Akiyoshi [Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu, E-mail: tanaka@sci.hokudai.ac.jp [Hokkaido University, Sapporo 060-0810 (Japan); Hokkaido University, Sapporo 060-0810 (Japan); Hirano, Nagisa [Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao; Yao, Min [Hokkaido University, Sapporo 060-0810 (Japan); Hokkaido University, Sapporo 060-0810 (Japan)

2013-11-01

Crystals of a member of the DING protein family (HPBP) were obtained accidentally, and the structure was determined at 1.35 Å resolution. For further analysis, a system for preparation of HPBP was constructed and the structure of a prepared sample was confirmed by X-ray crystal structure analysis at 1.03 Å resolution. After crystallization of a certain protein–RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.

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.

Crystal structure of Homo sapiens protein LOC79017

Energy Technology Data Exchange (ETDEWEB)

Bae, Euiyoung; Bingman, Craig A.; Aceti, David J.; Phillips, Jr., George N. (UW)

2010-02-08

LOC79017 (MW 21.0 kDa, residues 1-188) was annotated as a hypothetical protein encoded by Homo sapiens chromosome 7 open reading frame 24. It was selected as a target by the Center for Eukaryotic Structural Genomics (CESG) because it did not share more than 30% sequence identity with any protein for which the three-dimensional structure is known. The biological function of the protein has not been established yet. Parts of LOC79017 were identified as members of uncharacterized Pfam families (residues 1-95 as PB006073 and residues 104-180 as PB031696). BLAST searches revealed homologues of LOC79017 in many eukaryotes, but none of them have been functionally characterized. Here, we report the crystal structure of H. sapiens protein LOC79017 (UniGene code Hs.530024, UniProt code O75223, CESG target number go.35223).

ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

KAUST Repository

Hungler, Arnaud; Momin, Afaque Ahmad Imtiyaz; Diederichs, Kay; Arold, Stefan T.

2016-01-01

Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for 'crystallization and preliminary X-ray analysis' publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. A web server, titled ContaMiner, has been established, which allows fast molecular-replacement-based screening of crystallographic data against a database (ContaBase) of currently 62 potential contaminants. ContaMiner enables systematic screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for 'crystallization and preliminary X-ray analysis' publications. © Arnaud Hungler et al. 2016.

ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

KAUST Repository

Hungler, Arnaud

2016-11-02

Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for \\'crystallization and preliminary X-ray analysis\\' publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. A web server, titled ContaMiner, has been established, which allows fast molecular-replacement-based screening of crystallographic data against a database (ContaBase) of currently 62 potential contaminants. ContaMiner enables systematic screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for \\'crystallization and preliminary X-ray analysis\\' publications. © Arnaud Hungler et al. 2016.

Crystallization of the A-Domain of the Mannitol Transport Protein Enzyme IImtl

NARCIS (Netherlands)

Lammers, Leidy A.; Dijkstra, Bauke W.; Weeghel, Rob P. van; Pas, Hendri H.; Robillard, George T.

1992-01-01

The A-domain of the mannitol transport protein enzyme IImtl from Escherichia coli (relative molecular mass 16,300) was crystallized, both at room temperature and 4°C, from 40% polyethylene glycol 6000 (pH 8.5 to 9.0) using the hanging-drop method of vapour diffusion. The crystals have the monoclinic

Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

Directory of Open Access Journals (Sweden)

Morten K. Grøftehauge

2015-07-01

Full Text Available As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

Energy Technology Data Exchange (ETDEWEB)

Molina, Rafael [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); González, Ana; Moscoso, Miriam; García, Pedro [Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Stelter, Meike; Kahn, Richard [Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF, Laboratoire de Cristallographie Macromoléculaire, 41 Rue Jules Horowitz, 38027 Grenoble CEDEX 1 (France); Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain)

2007-09-01

The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L{sub III} absorption edge using synchrotron radiation.

Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae

International Nuclear Information System (INIS)

Molina, Rafael; González, Ana; Moscoso, Miriam; García, Pedro; Stelter, Meike; Kahn, Richard; Hermoso, Juan A.

2007-01-01

The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution. Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P2 1 2 1 2, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L III absorption edge using synchrotron radiation

Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

International Nuclear Information System (INIS)

Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

2006-01-01

The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

C-Reactive Protein Binds to Cholesterol Crystals and Co-Localizes with the Terminal Complement Complex in Human Atherosclerotic Plaques

DEFF Research Database (Denmark)

Pilely, Katrine; Fumagalli, Stefano; Rosbjerg, Anne

2017-01-01

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are seru...

Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein

International Nuclear Information System (INIS)

Hakim, Aaron; Thakral, Durga; Zhu, Darren F.; Nguyen, Jennifer B.

2012-01-01

A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution. Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = b = 46.46, c = 193.21 Å

Crystal Structure Analysis and the Identification of Distinctive Functional Regions of the Protein Elicitor Mohrip2.

Science.gov (United States)

Liu, Mengjie; Duan, Liangwei; Wang, Meifang; Zeng, Hongmei; Liu, Xinqi; Qiu, Dewen

2016-01-01

The protein elicitor MoHrip2, which was extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, the detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we investigated the structure of MoHrip2 to elucidate its functions based on molecular structure. The three-dimensional structure of MoHrip2 was obtained. Overall, the crystal structure formed a β-barrel structure and showed high similarity to the pathogenesis-related (PR) thaumatin superfamily protein thaumatin-like xylanase inhibitor (TL-XI). To investigate the functional regions responsible for MoHrip2 elicitor activities, the full length and eight truncated proteins were expressed in Escherichia coli and were evaluated for elicitor activity in tobacco. Biological function analysis showed that MoHrip2 triggered the defense system against Botrytis cinerea in tobacco. Moreover, only MoHrip2M14 and other fragments containing the 14 amino acids residues in the middle region of the protein showed the elicitor activity of inducing a hypersensitive response and resistance related pathways, which were similar to that of full-length MoHrip2. These results revealed that the central 14 amino acid residues were essential for anti-pathogenic activity.

Crystal Structure Analysis and the Identification of Distinctive Functional Regions of the Protein Elicitor Mohrip2

Directory of Open Access Journals (Sweden)

Mengjie Liu

2016-07-01

Full Text Available The protein elicitor MoHrip2, which was extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, the detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we investigated the structure of MoHrip2 to elucidate its functions based on molecular structure. The 3-dimensional structure of MoHrip2 was obtained. Overall, the crystal structure formed a β-barrel structure and showed high similarity to the pathogenesis-related (PR thaumatin superfamily protein thaumatin-like xylanase inhibitor (TL-XI. To investigate the functional regions responsible for MoHrip2 elicitor activities, the full length and 8 truncated proteins were expressed in Escherichia coli and were evaluated for elicitor activity in tobacco. Biological function analysis showed that MoHrip2 triggered the defense system against Botrytis cinerea in tobacco. Moreover, only MoHrip2M14 and other fragments containing the 14 amino acids residues in the middle region of the protein showed the elicitor activity of inducing a hypersensitive response and resistance related pathways, which were similar to that of full-length MoHrip2. These results revealed that the central 14 amino acid residues were essential for anti-pathogenic activity.

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

Science.gov (United States)

Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

2017-11-01

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

International Nuclear Information System (INIS)

Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

2007-01-01

A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3 1 21 or P3 2 21

Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

International Nuclear Information System (INIS)

Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

2006-01-01

The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P2 1 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively. The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2 1 , with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2 1 and R32, respectively

Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

Energy Technology Data Exchange (ETDEWEB)

Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

2012-06-22

Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

International Nuclear Information System (INIS)

Tang, Xuhua; Hew, Choy Leong

2007-01-01

The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2 1 2 1 2 1 , with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution

Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

Energy Technology Data Exchange (ETDEWEB)

Tang, Xuhua; Hew, Choy Leong, E-mail: dbshewcl@nus.edu.sg [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore)

2007-07-01

The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

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.

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

Crystallization of a 79 kDa fragment of the hook protein FlgE from Campylobacter jejuni

International Nuclear Information System (INIS)

Kido, Yasuji; Yoon, Young-Ho; Samatey, Fadel A.

2011-01-01

A 79 kDa fragment of FlgE from C. jejuni has been crystallized. A 79 kDa fragment of the bacterial flagellar hook protein FlgE from Campylobacter jejuni was cloned, overexpressed, purified and crystallized. Two different crystal forms were obtained. Synchrotron X-ray diffraction data showed that the first crystal form, which diffracted to 4.9 Å resolution, belonged to the tetragonal crystal system, with space group I4 1 22 and unit-cell parameters a = b = 186.2, c = 386.6 Å, α = β = γ = 90°. The second crystal form diffracted to 2.5 Å resolution and belonged to the monoclinic crystal system, with space group P2 1 and unit-cell parameters a = 75.7, b = 173.8, c = 150.8 Å, α = γ = 90, β = 106.5°. SeMet protein was also overexpressed, purified and crystallized, and a 2.6 Å resolution MAD data set was collected

Computer simulations of radiation damage in protein crystals; Simulationsrechnungen zu Strahlenschaeden an Proteinkristallen

Energy Technology Data Exchange (ETDEWEB)

Zehnder, M

2007-03-15

The achievable resolution and the quality of the dataset of an intensity data collection for structure analysis of protein crystals with X-rays is limited among other factors by radiation damage. The aim of this work is to obtain a better quantitative understanding of the radiation damage process in proteins. Since radiation damage is unavoidable it was intended to look for the optimum ratio between elastically scattered intensity and radiation damage. Using a Monte Carlo algorithm physical processes after an inelastic photon interaction are studied. The main radiation damage consists of ionizations of the atoms through the electron cascade following any inelastic photon interaction. Results of the method introduced in this investigation and results of an earlier theoretical studies of the influence of Auger-electron transport in diamond are in a good agreement. The dependence of the radiation damage as a function of the energy of the incident photon was studied by computer-aided simulations. The optimum energy range for diffraction experiments on the protein myoglobin is 10-40 keV. Studies of radiation damage as a function of crystal volume and shape revealed that very small plate or rod shaped crystals suffer less damage than crystals formed like a cube with the same volume. Furthermore the influence of a few heavy atoms in the protein molecule on radiation damage was examined. Already two iron atoms in the unit cell of myoglobin increase radiation damage significantly. (orig.)

Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

NARCIS (Netherlands)

Olijve, L.L.C.; Meister, K.; DeVries, A.L.; Duman, J.G.; Guo, S.; Bakker, H.J.; Voets, I.K.

2016-01-01

Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of

Crystal Structure of VC0702 at 2.0 Angstrom: Conserved Hypothetical Protein from Vibrio Cholerae

International Nuclear Information System (INIS)

Ni, S.; Forouhar, F.; Bussiere, D.; Robinson, H.; Kennedy, M.

2006-01-01

VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 Angstroms and refined to R work = 22.8% and R free = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a = 66.61 Angstroms, b = 88.118 Angstroms, and c = 118.35 Angstroms with a homodimer in the asymmetric unit. VC0702, which forms a mixed α + β three-layered αβα sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

Directory of Open Access Journals (Sweden)

Nusrat Yasmin

2017-01-01

Full Text Available A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+ and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Co-ordinated functions of Mms proteins define the surface structure of cubo-octahedral magnetite crystals in magnetotactic bacteria.

Science.gov (United States)

Arakaki, Atsushi; Yamagishi, Ayana; Fukuyo, Ayumi; Tanaka, Masayoshi; Matsunaga, Tadashi

2014-08-01

Magnetotactic bacteria synthesize magnetosomes comprised of membrane-enveloped single crystalline magnetite (Fe3 O4 ). The size and morphology of the nano-sized magnetite crystals (Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo-octahedral magnetite crystals in Magnetospirillum magneticum strain AMB-1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co-ordinated functions of Mms proteins regulate the morphology of the cubo-octahedral magnetite crystals in magnetotactic bacteria. © 2014 John Wiley & Sons Ltd.

In vitro uptake of 140 kDa Bacillus thuringiensis nematicidal crystal proteins by the second stage juvenile of Meloidogyne hapla.

Directory of Open Access Journals (Sweden)

Fengjuan Zhang

Full Text Available Plant-parasitic nematodes (PPNs are piercing/sucking pests, which cause severe damage to crops worldwide, and are difficult to control. The cyst and root-knot nematodes (RKN are sedentary endoparasites that develop specialized multinucleate feeding structures from the plant cells called syncytia or giant cells respectively. Within these structures the nematodes produce feeding tubes, which act as molecular sieves with exclusion limits. For example, Heterodera schachtii is reportedly unable to ingest proteins larger than 28 kDa. However, it is unknown yet what is the molecular exclusion limit of the Meloidogyne hapla. Several types of Bacillus thuringiensis crystal proteins showed toxicity to M. hapla. To monitor the entry pathway of crystal proteins into M. hapla, second-stage juveniles (J2 were treated with NHS-rhodamine labeled nematicidal crystal proteins (Cry55Aa, Cry6Aa, and Cry5Ba. Confocal microscopic observation showed that these crystal proteins were initially detected in the stylet and esophageal lumen, and subsequently in the gut. Western blot analysis revealed that these crystal proteins were modified to different molecular sizes after being ingested. The uptake efficiency of the crystal proteins by the M. hapla J2 decreased with increasing of protein molecular mass, based on enzyme-linked immunosorbent assay analysis. Our discovery revealed 140 kDa nematicidal crystal proteins entered M. hapla J2 via the stylet, and it has important implications in designing a transgenic resistance approach to control RKN.

High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method

International Nuclear Information System (INIS)

Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

2013-01-01

Using the high-pressure cryocooling method, the high-resolution X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. This is the first ultra-high-resolution structure obtained from a high-pressure cryocooled crystal. Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005 ▶) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method

Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles

International Nuclear Information System (INIS)

Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

2013-01-01

Novel hepatitis B virus-like particles of recombinant dimeric core–GFP fusion protein were expressed, purified and crystallized. The crystals diffracted to 2.15 Å resolution and belonged to space group F432, with unit-cell parameters a = b = c = 219.7 Å. Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions

Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus

International Nuclear Information System (INIS)

Shang, Guijun; Cang, Huaixing; Liu, Zhijie; Gao, Wei; Bi, Ruchang

2010-01-01

Recombinant calcineurin B-like protein 1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K + , osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298 K with PEG 2000 MME as a precipitant diffracted to 2.90 Å resolution. The crystal belonged to space group P2 1 2 1 2, with unit-cell parameters a = 99.87, b = 114.42, c = 63.80 Å, α = β = γ = 90.00° and three molecules per asymmetric unit

Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

DEFF Research Database (Denmark)

Guerra, B; Niefind, K; Pinna, L A

1998-01-01

The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

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

Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

International Nuclear Information System (INIS)

Delfosse, Vanessa; Hugonnet, Jean-Emmanuel; Sougakoff, Wladimir; Mayer, Claudine

2005-01-01

The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported. The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry

Purification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum).

Science.gov (United States)

Sharma, Urvashi; Suresh, C G

2011-06-01

A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS-PAGE. The IC(50) value of CPTI was determined to be 2.5 µg against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2(1)2(1)2 form A, P2(1)2(1)2 form B and P2(1)2(1)2(1). The crystals of P2(1)2(1)2 form A, with unit-cell parameters a = 37.2, b = 41.2, c = 104.6 Å, diffracted to 2.0 Å resolution at the home source and to 1.4 Å on beamline BM14 at the ESRF. Data were also collected from crystals grown in the presence of iodine. The Matthews coefficient for these crystals was calculated to be 2.37 Å(3) Da(-1), corresponding to a solvent content of 42%. The other two crystal forms (P2(1)2(1)2 form B and P2(1)2(1)2(1)) diffracted comparatively poorly.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114

International Nuclear Information System (INIS)

Martínez-Rodríguez, Sergio; González-Ramírez, Luis Antonio; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier; Gavira, Jose Antonio; García-Ruiz, Juan Ma.

2007-01-01

Crystals of an active-site mutated hydantoin racemase from S. meliloti have been obtained in the presence and absence of d,l-5-isopropyl-hydantoin and characterized by X-ray diffraction. A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å, crystal volumes per protein weight (V M ) of 1.94 and 1.98 Å 3 Da −1 and solvent contents of 36.7 and 37.9%, respectively

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

DEFF Research Database (Denmark)

Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

2007-01-01

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

Crystallization, data collection and phasing of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri

International Nuclear Information System (INIS)

Santacruz, C. P.; Balan, A.; Ferreira, L. C. S.; Barbosa, J. A. R. G.

2006-01-01

The molybdate-binding protein (ModA) from X. axonopodis pv. citri was crystallized with sodium molybdate in the presence of PEG or sulfate. The crystal diffracted to a maximum resolution of 1.7 Å and belongs to the orthorhombic space group C222 1, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 Å using synchrotron radiation. The crystal belongs to the orthorhombic space group C222 1 , with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress

Crystallization, data collection and phasing of the molybdate-binding protein of the phytopathogen Xanthomonas axonopodis pv. citri

Energy Technology Data Exchange (ETDEWEB)

Santacruz, C. P.; Balan, A.; Ferreira, L. C. S. [Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, São Paulo, SP (Brazil); Barbosa, J. A. R. G., E-mail: joao@lnls.br [Centro de Biologia Molecular e Estrutural (CeBiMe), Laboratório Nacional de Luz Síncrotron (LNLS), CP 6192, Campinas, SP 13084-971 (Brazil); Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, São Paulo, SP (Brazil)

2006-03-01

The molybdate-binding protein (ModA) from X. axonopodis pv. citri was crystallized with sodium molybdate in the presence of PEG or sulfate. The crystal diffracted to a maximum resolution of 1.7 Å and belongs to the orthorhombic space group C222{sub 1,} with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. Xanthomonas axonopodis pv. citri ModA protein is the ABC periplasmic binding component responsible for the capture of molybdate. The protein was crystallized with sodium molybdate using the hanging-drop vapour-diffusion method in the presence of PEG or sulfate. X-ray diffraction data were collected to a maximum resolution of 1.7 Å using synchrotron radiation. The crystal belongs to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 68.15, b = 172.14, c = 112.04 Å. The crystal structure was solved by molecular-replacement methods and structure refinement is in progress.

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

International Nuclear Information System (INIS)

Rantanen, Mika K.; Lehtiö, Lari; Rajagopal, Lakshmi; Rubens, Craig E.; Goldman, Adrian

2006-01-01

Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

Energy Technology Data Exchange (ETDEWEB)

Rantanen, Mika K.; Lehtiö, Lari [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland); Rajagopal, Lakshmi; Rubens, Craig E. [Division of Infectious Disease, Children’s Hospital and Regional Medical Center, Seattle, Washington 98105 (United States); Goldman, Adrian, E-mail: adrian.goldman@helsinki.fi [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland)

2006-09-01

Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

Science.gov (United States)

Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

2008-06-01

Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

International Nuclear Information System (INIS)

Linke, Christian; Caradoc-Davies, Tom T.; Proft, Thomas; Baker, Edward N.

2008-01-01

The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution

Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

Energy Technology Data Exchange (ETDEWEB)

Linke, Christian, E-mail: clin180@ec.auckland.ac.nz [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Caradoc-Davies, Tom T. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Australian Synchrotron, Clayton, Victoria 3168 (Australia); Proft, Thomas [School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Baker, Edward N. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand)

2008-02-01

The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.

Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

Science.gov (United States)

Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

2016-09-16

Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums. Copyright © 2016 Elsevier Inc. All rights reserved.

Crystallization and preliminary electron diffraction study to 3. 7 A of DNA helix-destabilizing protein gp32*I

Energy Technology Data Exchange (ETDEWEB)

Chiu, W; Hosoda, J

1978-01-01

A two-dimensionally large and thin crystal has been obtained from gp32*I, a proteolytically digested product of a DNA helix-destabilizing protein coded by gene 32 in bacteriophage T4. High-resolution electron diffraction patterns (approx. 3.7 A) are recorded from both unstained and stained protein crystals embedded in glucose. The crystal is of orthorhombic space group with a = 62.9 A and b = 47.3 A.

NMR structure of the protein NP-247299.1: comparison with the crystal structure

International Nuclear Information System (INIS)

Jaudzems, Kristaps; Geralt, Michael; Serrano, Pedro; Mohanty, Biswaranjan; Horst, Reto; Pedrini, Bill; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

2010-01-01

Comparison of the NMR and crystal structures of a protein determined using largely automated methods has enabled the interpretation of local differences in the highly similar structures. These differences are found in segments of higher B values in the crystal and correlate with dynamic processes on the NMR chemical shift timescale observed in solution. The NMR structure of the protein NP-247299.1 in solution at 313 K has been determined and is compared with the X-ray crystal structure, which was also solved in the Joint Center for Structural Genomics (JCSG) at 100 K and at 1.7 Å resolution. Both structures were obtained using the current largely automated crystallographic and solution NMR methods used by the JCSG. This paper assesses the accuracy and precision of the results from these recently established automated approaches, aiming for quantitative statements about the location of structure variations that may arise from either one of the methods used or from the different environments in solution and in the crystal. To evaluate the possible impact of the different software used for the crystallographic and the NMR structure determinations and analysis, the concept is introduced of reference structures, which are computed using the NMR software with input of upper-limit distance constraints derived from the molecular models representing the results of the two structure determinations. The use of this new approach is explored to quantify global differences that arise from the different methods of structure determination and analysis versus those that represent interesting local variations or dynamics. The near-identity of the protein core in the NMR and crystal structures thus provided a basis for the identification of complementary information from the two different methods. It was thus observed that locally increased crystallographic B values correlate with dynamic structural polymorphisms in solution, including that the solution state of the protein involves

On the behavior of water at subfreezing temperatures in a protein crystal: evidence of higher mobility than in bulk water.

Science.gov (United States)

Wang, Dongqi; Böckmann, Anja; Dolenc, Jožica; Meier, Beat H; van Gunsteren, Wilfred F

2013-10-03

NMR experiments have shown that water molecules in the crystal of the protein Crh are still mobile at temperatures well below 273 K. In order to investigate this water anomaly, a molecular dynamics (MD) simulation study of crystalline Crh was carried out to determine the mobility of water in this crystal. The simulations were carried out at three temperatures, 150, 200, and 291 K. Simulations of bulk water at these temperatures were also done to obtain the properties of the simple point charge (SPC) water model used at these temperatures and to allow a comparison of the properties of water in the Crh crystal with those of bulk water at the same temperatures. According to the simulations, water is immobilized at 150 K both in crystal and in bulk water. As expected, at 291 K it diffuses and rotates more slowly in the protein crystal than in bulk water. However, at 200 K, the translational and rotational mobility of the water molecules is larger in the crystal than in bulk water. The enhancement of water mobility in the crystal at 200 K was further investigated by MD simulations in which the backbone or all protein atoms were positionally restrained, and in which additionally the electrostatic protein-water interactions were removed. Of these changes in the environment of the water molecules, rigidifying the protein backbones slightly enhanced water diffusion, while it slowed down rotation. In contrast, removal of electrostatic protein-water interactions did not change water diffusion but enhanced rotational motion significantly. Further investigations are required to delineate particular features of the protein crystal that induce the anomalous behavior of water at 200 K.

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

The influence of magnetic fields on protein crystal growth and quality; Zum Einfluss magnetischer Felder auf das Wachstum und die Qualitaet von Proteinkristallen

Energy Technology Data Exchange (ETDEWEB)

Meents, Alke

2005-08-01

Magnetic fields can affect protein crystal growth in several ways. In homogeneous magnetic fields molecules and crystallites line up themselves along the magnetic field direction due to their magnetic anisotropy. Inhomogeneous magnetic fields exert a force on diamagnetic and paramagnetic compounds towards regions of lower or higher field strength. This effect can be used to create a microgravity-like environment for diamagnetic proteins and an environment comparable to hypergravity for paramagnetic proteins. Crystallization in homogeneous magnetic fields and a microgravity-like environment are reported to have a positive effect on crystal quality. The aim of this work was to systematically investigate the effect of protein crystallization in magnetic fields on the crystal quality by comparing a large number of crystals grown under identical conditions with- and without magnetic fields. Crystal quality was determined by means of high resolution rocking-curve measurements. Furthermore in certain cases complete diffraction datasets were collected. Any possible influence of magnetic fields on the mosaicity and the quality of the diffraction data was evaluated statistically by applying Wilcoxon-Ranksum tests. To investigate the effect of protein crystallization in homogeneous magnetic fields the diamagnetic proteins Thaumatin, Trypsin, and Lysozyme and paramagnetic Myoglobin were crystallized in magnetic fields of 5 T, 8.8 T, and 15.8 T. The analysis of crystal mosaicity and quality of the diffraction data of the diamagnetic proteins did not reveal a significant influence on the crystal quality. In contrast the crystals of paramagnetic Myoglobin grew up to 14 times larger than the ones in the control experiment. In addition they had a significant lower mosaicity, and diffracted to a higher resolution than ever reported before. Special pole pieces for an existing magnet were designed and build to grow protein crystals in an inhomogeneous magnetic field The experimental

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Science.gov (United States)

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

Science.gov (United States)

Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

2012-10-09

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Crystal structure of the Japanese encephalitis virus envelope protein.

Science.gov (United States)

Luca, Vincent C; AbiMansour, Jad; Nelson, Christopher A; Fremont, Daved H

2012-02-01

Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na+/H+ exchanger NHE1

International Nuclear Information System (INIS)

Ben Ammar, Youssef; Takeda, Soichi; Sugawara, Mitsuaki; Miyano, Masashi; Mori, Hidezo; Wakabayashi, Shigeo

2005-01-01

Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca 2+ -binding protein that directly interacts with and regulates the activity of all plasma-membrane Na + /H + -exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å

Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures

Science.gov (United States)

2014-01-01

We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5–22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10–25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function. PMID:24392845

Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution

DEFF Research Database (Denmark)

Niefind, K; Guerra, B; Pinna, L A

1998-01-01

CK2alpha is the catalytic subunit of protein kinase CK2, an acidophilic and constitutively active eukaryotic Ser/Thr kinase involved in cell proliferation. A crystal structure, at 2.1 A resolution, of recombinant maize CK2alpha (rmCK2alpha) in the presence of ATP and Mg2+, shows the enzyme in an ...

Protein Adsorption to Titanium and Zirconia Using a Quartz Crystal Microbalance Method

Directory of Open Access Journals (Sweden)

You Kusakawa

2017-01-01

Full Text Available Protein adsorption onto titanium (Ti or zirconia (ZrO2 was evaluated using a 27 MHz quartz crystal microbalance (QCM. As proteins, fibronectin (Fn, a cell adhesive protein, and albumin (Alb, a cell adhesion-inhibiting protein, were evaluated. The Ti and ZrO2 sensors for QCM were characterized by atomic force microscopy and electron probe microanalysis observation, measurement of contact angle against water, and surface roughness. The amounts of Fn and Alb adsorbed onto the Ti and ZrO2 sensors and apparent reaction rate were obtained using QCM measurements. Ti sensor showed greater adsorption of Fn and Alb than the ZrO2 sensor. In addition, amount of Fn adsorbed onto the Ti or ZrO2 sensors was higher than that of Alb. The surface roughness and hydrophilicity of Ti or ZrO2 may influence the adsorption of Fn or Alb. With regard to the adsorption rate, Alb adsorbed more rapidly than Fn onto Ti. Comparing Ti and ZrO2, Alb adsorption rate to Ti was faster than that to ZrO2. Fn adsorption will be effective for cell activities, but Alb adsorption will not. QCM method could simulate in vivo Fn and Alb adsorption to Ti or ZrO2.

Some Aspects of Crystal Centering During X-ray High-throughput Protein Crystallography Experiment

Science.gov (United States)

Gaponov, Yu. A.; Matsugaki, N.; Sasajima, K.; Igarashi, N.; Wakatsuki, S.

A set of algorithms and procedures of a crystal loop centering during X-ray high-throughput protein crystallography experiment has been designed and developed. A simple algorithm of the crystal loop detection and preliminary recognition has been designed and developed. The crystal loop detection algorithm is based on finding out the crystal loop ending point (opposite to the crystal loop pin) using image cross section (digital image column) profile analysis. The crystal loop preliminary recognition procedure is based on finding out the crystal loop sizes and position using image cross section profile analysis. The crystal loop fine recognition procedure based on Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed. The procedure of restoring missing coordinate of the crystal loop is described. Based on developed algorithms and procedures the optimal auto-centering procedure has been designed and developed. A procedure of optimal manual crystal centering (Two Clicks Procedure) has been designed and developed. Developed procedures have been integrated into control software system PCCS installed at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK.

Imaging transport phenomena during lysozyme protein crystal growth by the hanging drop technique

Science.gov (United States)

Sethia Gupta, Anamika; Gupta, Rajive; Panigrahi, P. K.; Muralidhar, K.

2013-06-01

The present study reports the transport process that occurs during the growth of lysozyme protein crystals by the hanging drop technique. A rainbow schlieren technique has been employed for imaging changes in salt concentration. A one dimensional color filter is used to record the deflection of the light beam. An optical microscope and an X-ray crystallography unit are used to characterize the size, tetragonal shape and Bravais lattice constants of the grown crystals. A parametric study on the effect of drop composition, drop size, reservoir height and number of drops on the crystal size and quality is reported. Changes in refractive index are not large enough to create a meaningful schlieren image in the air gap between the drop and the reservoir. However, condensation of fresh water over the reservoir solution creates large changes in the concentration of NaCl, giving rise to clear color patterns in the schlieren images. These have been analyzed to obtain salt concentration profiles near the free surface of the reservoir solution as a function of time. The diffusion of fresh water into the reservoir solution at the early stages of crystal growth followed by the mass flux of salt from the bulk solution towards the free surface has been recorded. The overall crystal growth process can be classified into two regimes, as demarcated by the changes in slope of salt concentration within the reservoir. The salt concentration in the reservoir equilibrates at long times when the crystallization process is complete. Thus, transport processes in the reservoir emerge as the route to monitor protein crystal growth in the hanging drop configuration. Results show that crystal growth rate is faster for a higher lysozyme concentration, smaller drops, and larger reservoir heights.

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

International Nuclear Information System (INIS)

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

2008-01-01

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

The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

International Nuclear Information System (INIS)

Smith, C.A.; Cohen, A.E.

2009-01-01

The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.

Expression, purification, crystallization and preliminary crystallographic analysis of laminin-binding protein (Lmb) from Streptococcus agalactiae

International Nuclear Information System (INIS)

Ragunathan, Preethi; Spellerberg, Barbara; Ponnuraj, Karthe

2009-01-01

Laminin-binding protein from S. agalactiae was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution. Laminin-binding protein (Lmb), a surface-exposed lipoprotein from Streptococcus agalactiae (group B streptococcus), mediates attachment to human laminin and plays a crucial role in the adhesion/invasion of eukaryotic host cells. However, the structural basis of laminin binding still remains unclear. In the context of detailed structural analysis, the lmb gene has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals diffracted to a resolution of 2.5 Å and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 56.63, b = 70.60, c = 75.37 Å, β = 96.77°

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

Science.gov (United States)

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

2017-11-01

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

Crystallization of the novel flavodoxine-like protein, WrbA, - on the way to three-dimensional structure

Czech Academy of Sciences Publication Activity Database

Kutá-Smatanová, Ivana; Wolfová, J.; Grandori, R.; Chatterjee, N.; Carey, J.

2004-01-01

Roč. 54, č. 2004 (2004), s. 35 ISSN 1210-8529. [European Crystallographic Meeting /22./. Budapešť, 26.08.2004-31.08.2004] R&D Projects: GA MŠk ME 640; GA MŠk LN00A141 Grant - others:GA ÚV1 INT-03-09049 Keywords : flavodoxine-like protein, tryprophan-repressor binding protein, WrbA apoprotein, WrbA holoprotein, FMN, standard crystallization techniques, advanced crystallization techniques Subject RIV: BO - Biophysics

PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

Directory of Open Access Journals (Sweden)

Huilin Wang

Full Text Available X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed 'PredPPCrys' using the support vector machine (SVM. Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I. Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II, which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization

Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G-Proteins, PIP2 and Sodium

Science.gov (United States)

Whorton, Matthew R.; MacKinnon, Roderick

2011-01-01

Summary G-protein-gated K+ channels (Kir3.1–Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating. PMID:21962516

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

Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins

International Nuclear Information System (INIS)

Renzi, Fabiana; Panetta, Gianna; Vallone, Beatrice; Brunori, Maurizio; Arceci, Massimo; Bozzoni, Irene; Laneve, Pietro; Caffarelli, Elisa

2006-01-01

Recombinant His-tagged XendoU, a eukaryotic endoribonuclease, appeared to aggregate in the presence of divalent cations. Monodisperse protein which yielded crystals diffracting to 2.2 Å was obtained by addition of EDTA. XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3 1 21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution

Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

International Nuclear Information System (INIS)

Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

2009-01-01

Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

Effects of insecticidal crystal proteins (Cry proteins) produced by genetically modified maize (Bt maize) on the nematode Caenorhabditis elegans

International Nuclear Information System (INIS)

Höss, Sebastian; Menzel, Ralph; Gessler, Frank; Nguyen, Hang T.; Jehle, Johannes A.; Traunspurger, Walter

2013-01-01

The genetically modified maize MON89034 × MON88017 expresses different crystal (Cry) proteins with pesticidal activity against the European corn borer (Cry1.105; Cry2Ab2) and the Western corn root worm (Cry3Bb1). Non-target organisms, such as soil nematodes, might be exposed to the Cry proteins that enter the soil in course of crop growing. Therefore, the risk of those proteins for nematodes was assessed by testing their toxic effects on Caenorhabditis elegans. All three insecticidal Cry proteins showed dose-dependent inhibitory effects on C. elegans reproduction (EC50: 0.12–0.38 μmol L −1 ), however, at concentrations that were far above the expected soil concentrations. Moreover, a reduced toxicity was observed when Cry proteins were added jointly. A C. elegans mutant strain deficient for receptors for the nematicidal Cry5B was also resistant against Cry1.105 and Cry2Ab2, suggesting that these Cry proteins bound to the same or similar receptors as nematicidal Cry proteins and thereby affect the reproduction of C. elegans. -- Highlights: •Insecticidal Cry proteins dose-dependently inhibited the reproduction of C. elegans. •Mixture toxicity was lower than expected from concentration-additive single effects. •Genes for MAPK-defense-pathway were up-regulated in presence of Cry protein mixture. •Knock-out strains deficient for Cry5B-receptors showed lower susceptibility to insecticidal Cry proteins. •Toxicity of insecticidal Cry-proteins on C. elegans occurred at concentrations far above expected field concentrations. -- Insecticidal Cry proteins expressed by genetically modified maize act on nematodes via a similar mode of action as nematicidal Cry proteins, however, at concentrations far above expected soil levels

Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein

International Nuclear Information System (INIS)

Ito, Sohei; Tatsuda, Emi; Ishino, Kousuke; Suzuki, Kenichiro; Sakai, Hiroshi; Uchida, Koji

2006-01-01

Antigen-free Fab fragment of mAbR310, which recognizes (R)-HNE modified protein, has been crystallized. Initial phases have been obtained by molecular replacement. 4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE–histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNE-modified protein, are reported. The Fab fragment of mAbR310 was obtained by digestion with papain, purified and crystallized. Using hanging-drop vapour-diffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 Å, β = 118.88°) and diffracted X-rays to a resolution of 1.84 Å. The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 Å 3 Da −1 and a solvent content of 51.0%

Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster

Energy Technology Data Exchange (ETDEWEB)

Hoffmann, Anita; Neumann, Piotr [Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Schierhorn, Angelika [Max-Planck-Institut für Proteinfaltung, Abteilung Massenspektrometrie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de [Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany); Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine (MZP), Martin-Luther-Universität Halle-Wittenberg (Germany)

2008-08-01

Crystallization of the cystine-knot protein Spätzle occurred following serendipitous limited degradation of the pro-Spätzle propeptide during the crystallization experiment. The Spätzle protein is involved in both the definition of the dorsal–ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 Å resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 Å) that diffracted to 2.8 Å resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.

Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

Science.gov (United States)

Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

2013-06-24

Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability

International Nuclear Information System (INIS)

Zhu, W.; Manjasetty, B.; Chance, M.

2007-01-01

The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn 2+ for its catalytic activity. Crystals of the ECAI + Mn 2+ complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn 2+ complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 (angstrom) resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn 2+ ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structural features that are of importance to the intrinsic activity and heat stability of AI

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°

Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

Energy Technology Data Exchange (ETDEWEB)

Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

2015-03-05

Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

[Genes of insecticidal crystal proteins with dual specificity in Bacillus thuringiensis strains, isolated in the Crimea territory].

Science.gov (United States)

Rymar, S Iu; Isakova, I A; Kuznietsova, L M; Kordium, V A

2006-01-01

The insecticidal crystal proteins of 15 B. thuringiensis strains, isolated in the Crimea territory that are toxical for some Lepidoptera and Colorado potato beetle larvae were identified by PAGE electrophoresis. Ten strains produced the crystal proteins with high molecular weight (> 120 kD). PCR with use of broad specificity primers and DNA of these B. thuringiensis strains as template demonstrated the specific PCR products (1000 bp). Amplified DNA fragments were cloned and sequenced. The nucleotide sequence analysis revealed that the genomes of ten strains of B. thuringiensis carried Cry1B genes, which are responsible for production of the insecticidal crystal proteins with dual specificity. The influence of the solubilization conditions on the structure and toxicity of Cry1B protein for Colorado potato beetle larvae was shown. The dual toxicity of studied B. thuringiensis strains is explained by the Cry1B genes presence in their genomes. These strains may be used to develop the broad specificity bioinsecticides.

A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

International Nuclear Information System (INIS)

Nederlof, Igor; Genderen, Eric van; Li, Yao-Wang; Abrahams, Jan Pieter

2013-01-01

An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA. When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e − Å −2 ), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

Science.gov (United States)

Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

2017-08-08

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

OpenAIRE

Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

2013-01-01

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolec...

Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na{sup +}/H{sup +} exchanger NHE1

Energy Technology Data Exchange (ETDEWEB)

Ben Ammar, Youssef [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Takeda, Soichi [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Sugawara, Mitsuaki; Miyano, Masashi [Structural Biophysics Laboratory, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki, Sayo, Hyogo 679-5148 (Japan); Mori, Hidezo [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Wakabayashi, Shigeo, E-mail: wak@ri.ncvc.go.jp [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan)

2005-10-01

Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca{sup 2+}-binding protein that directly interacts with and regulates the activity of all plasma-membrane Na{sup +}/H{sup +}-exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å.

Two crystal forms of a helix-rich fatty acid- and retinol-binding protein, Na-FAR-1, from the parasitic nematode Necator americanus

International Nuclear Information System (INIS)

Gabrielsen, Mads; Rey-Burusco, M. Florencia; Griffiths, Kate; Roe, Andrew J.; Cooper, Alan; Smith, Brian O.; Kennedy, Malcolm W.; Corsico, Betina

2012-01-01

Na-FAR-1, a fatty acid- and retinol-binding protein, was expressed in bacteria, purified and crystallized. Crystals grew in two different morphologies under the same conditions. Na-FAR-1 is an unusual α-helix-rich fatty acid- and retinol-binding protein from Necator americanus, a blood-feeding intestinal parasitic nematode of humans. It belongs to the FAR protein family, which is unique to nematodes; no structural information is available to date for FAR proteins from parasites. Crystals were obtained with two different morphologies that corresponded to different space groups. Crystal form 1 exhibited space group P432 (unit-cell parameters a = b = c = 120.80 Å, α = β = γ = 90°) and diffracted to 2.5 Å resolution, whereas crystal form 2 exhibited space group F23 (unit-cell parameters a = b = c = 240.38 Å, α = β = γ = 90°) and diffracted to 3.2 Å resolution. Crystal form 2 showed signs of significant twinning

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

Expression, purification, crystallization and preliminary X-ray analysis of the human RuvB-like protein RuvBL1

International Nuclear Information System (INIS)

Gorynia, Sabine; Matias, Pedro M.; Gonçalves, Susana; Coelho, Ricardo; Lopes, Gonçalo; Thomaz, Mónica; Huber, Martina; Haendler, Bernard; Donner, Peter; Carrondo, Maria Arménia

2005-01-01

Human RuvB-like protein RuvBL1 plays important roles in essential signaling pathways like c-Myc and Wnt, in transcription, and in DNA repair and apoptosis. Crystals of both native and a Se-Met derivative were obtained and characterized. SAD data leading to the structure solution at 2.2 Å were measured from the Se-Met crystals. RuvBL1, an evolutionary highly conserved protein related to the AAA + family of ATPases, has been crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystals are hexagonal and belong to space group P6, with unit-cell parameters a = b = 207.1, c = 60.7 Å and three molecules in the asymmetric unit

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

International Nuclear Information System (INIS)

Souza, C. S.; Ferreira, L. C. S.; Thomas, L.; Barbosa, J. A. R. G.; Balan, A.

2009-01-01

The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6 1 22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 Å, and contained two molecules in the asymetric unit. It diffracted to 2.24 Å resolution

5,5'-Dithio-bis(2-nitrobenzoic acid) modification of cysteine improves the crystal quality of human chloride intracellular channel protein 2

International Nuclear Information System (INIS)

Mi Wei; Li Lanfen; Su Xiaodong

2008-01-01

Structural studies of human chloride intracellular channel protein 2 (CLIC2) had been hampered by the problem of generating suitable crystals primarily due to the protein containing exposed cysteines. Several chemical reagents were used to react with the cysteines on CLIC2 in order to modify the redox state of the protein. We have obtained high quality crystals that diffracted to better than 2.5 A at a home X-ray source by treating the protein with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). After solving the crystal structure of CLIC2, we found that the DTNB had reacted with the Cys 114 , and made CLIC2 in a homogenous oxidized state. This study demonstrated that the DTNB modification drastically improved the crystallization of CLIC2, and it implied that this method may be useful for other proteins containing exposed cysteines in general

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

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein from Stenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank.

Science.gov (United States)

Keegan, Ronan; Waterman, David G; Hopper, David J; Coates, Leighton; Taylor, Graham; Guo, Jingxu; Coker, Alun R; Erskine, Peter T; Wood, Steve P; Cooper, Jonathan B

2016-08-01

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) from Alcaligenes sp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement using MOLREP in which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in the MOLREP peak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. A BLAST search then indicated that the molecule was most probably a phosphate-binding protein from Stenotrophomonas maltophilia (UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of the S. maltophilia protein has been refined to an R factor of 10.15% and an Rfree of 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

NARCIS (Netherlands)

Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

2001-01-01

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

Energy Technology Data Exchange (ETDEWEB)

Upadhyay, Anup K.; Cyr, Matthew; Longenecker, Kenton; Tripathi, Rakesh; Sun, Chaohong; Kempf, Dale J. (AbbVie)

2017-02-21

The rapid spread of the recentZika virus(ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 ofZika virus(ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space groupP2₁2₁2 and containing two protein molecules in the asymmetric unit. The structure is similar to that reported for the NS5 protein fromJapanese encephalitis virusand suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.

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)

Laser scattering in a hanging drop vapor diffusion apparatus for protein crystal growth in a microgravity environment

Science.gov (United States)

Casay, G. A.; Wilson, W. W.

1992-01-01

One type of hardware used to grow protein crystals in the microgravity environment aboard the U.S. Space Shuttle is a hanging drop vapor diffusion apparatus (HDVDA). In order to optimize crystal growth conditions, dynamic control of the HDVDA is desirable. A critical component in the dynamically controlled system is a detector for protein nucleation. We have constructed a laser scattering detector for the HDVDA capable of detecting the nucleation stage. The detector was successfully tested for several scatterers differing in size using dynamic light scattering techniques. In addition, the ability to detect protein nucleation using the HDVDA was demonstrated for lysozyme.

High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins

Directory of Open Access Journals (Sweden)

Cha Sun-Shin

2007-05-01

Full Text Available Abstract Background Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1. Results The 1.6 Å crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im but some disparity to an immunity protein against carnobacteriocin B2 (ImB2 in both the C-terminal conformation and the local structure constructed by α3, α4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity. Conclusion The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.

A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

Energy Technology Data Exchange (ETDEWEB)

Nederlof, Igor; Genderen, Eric van; Li, Yao-Wang; Abrahams, Jan Pieter, E-mail: abrahams@chem.leidenuniv.nl [Leiden University, Einsteinweg 55, 2333 CC Leiden (Netherlands)

2013-07-01

An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA. When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e{sup −} Å{sup −2}), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

Comparison of NMR and crystal structures for the proteins TM1112 and TM1367

International Nuclear Information System (INIS)

Mohanty, Biswaranjan; Serrano, Pedro; Pedrini, Bill; Jaudzems, Kristaps; Geralt, Michael; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

2010-01-01

NMR structures of the proteins TM1112 and TM1367 solved by the JCSG in solution at 298 K could be superimposed with the corresponding crystal structures at 100 K with r.m.s.d. values of <1.0 Å for the backbone heavy atoms. For both proteins the structural differences between multiple molecules in the asymmetric unit of the crystals correlated with structural variations within the bundles of conformers used to represent the NMR solution structures. A recently introduced JCSG NMR structure-determination protocol, which makes use of the software package UNIO for extensive automation, was further evaluated by comparison of the TM1112 structure obtained using these automated methods with another NMR structure that was independently solved in another PSI center, where a largely interactive approach was applied. The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Å resolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of <1.0 Å for the backbone heavy atoms, which provided a starting platform to investigate local structure variations, which may arise from either the methods used or from the different chemical environments in solution and in the crystal. Thereby, these comparative studies were further explored with the use of reference NMR and crystal structures, which were computed using the NMR software with input of upper-limit distance constraints derived from the molecular models that represent the results of structure

Fluid-crystal coexistence for proteins and inorganic nanocolloids : Dependence on ionic strength

NARCIS (Netherlands)

Prinsen, P.; Odijk, T.

2006-01-01

We investigate theoretically the fluid-crystal coexistence of solutions of globular charged nanoparticles such as proteins and inorganic colloids. The thermodynamic properties of the fluid phase are computed via the optimized Baxter model P. Prinsen and T. Odijk [J. Chem. Phys. 121, 6525 (2004)].

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

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes

International Nuclear Information System (INIS)

Caffrey, Martin

2015-01-01

A comprehensive and up-to-date review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes is reported. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for

A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes

Energy Technology Data Exchange (ETDEWEB)

Caffrey, Martin, E-mail: martin.caffrey@tcd.ie [Trinity College Dublin, Dublin (Ireland)

2015-01-01

A comprehensive and up-to-date review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes is reported. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipid-anchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for

X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection.

Science.gov (United States)

Owen, Robin L; Yorke, Briony A; Pearson, Arwen R

2012-05-01

During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals. © 2012 International Union of Crystallography

Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

Science.gov (United States)

Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

2001-08-01

The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

Increasing the X-ray Diffraction Power of Protein Crystals by Dehydration: The Case of Bovine Serum Albumin and a Survey of Literature Data

Directory of Open Access Journals (Sweden)

Irene Russo Krauss

2012-03-01

Full Text Available Serum albumin is one of the most widely studied proteins. It is the most abundant protein in plasma with a typical concentration of 5 g/100 mL and the principal transporter of fatty acids in plasma. While the crystal structures of human serum albumin (HSA free and in complex with fatty acids, hemin, and local anesthetics have been characterized, no crystallographic models are available on bovine serum albumin (BSA, presumably because of the poor diffraction power of existing hexagonal BSA crystals. Here, the crystallization and diffraction data of a new BSA crystal form, obtained by the hanging drop method using MPEG 5K as precipitating agent, are presented. The crystals belong to space group C2, with unit-cell parameters a = 216.45 Å, b = 44.72 Å, c = 140.18 Å, β = 114.5°. Dehydration was found to increase the diffraction limit of BSA crystals from ~8 Å to 3.2 Å, probably by improving the packing of protein molecules in the crystal lattice. These results, together with a survey of more than 60 successful cases of protein crystal dehydration, confirm that it can be a useful procedure to be used in initial screening as a method of improving the diffraction limits of existing crystals.

Water polygons in high-resolution protein crystal structures.

Science.gov (United States)

Lee, Jonas; Kim, Sung-Hou

2009-07-01

We have analyzed the interstitial water (ISW) structures in 1500 protein crystal structures deposited in the Protein Data Bank that have greater than 1.5 A resolution with less than 90% sequence similarity with each other. We observed varieties of polygonal water structures composed of three to eight water molecules. These polygons may represent the time- and space-averaged structures of "stable" water oligomers present in liquid water, and their presence as well as relative population may be relevant in understanding physical properties of liquid water at a given temperature. On an average, 13% of ISWs are localized enough to be visible by X-ray diffraction. Of those, averages of 78% are water molecules in the first water layer on the protein surface. Of the localized ISWs beyond the first layer, almost half of them form water polygons such as trigons, tetragons, as well as expected pentagons, hexagons, higher polygons, partial dodecahedrons, and disordered networks. Most of the octagons and nanogons are formed by fusion of smaller polygons. The trigons are most commonly observed. We suggest that our observation provides an experimental basis for including these water polygon structures in correlating and predicting various water properties in liquid state.

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

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

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.

Crystallization and preliminary crystallographic studies of L30e, a ribosomal protein from Methanocaldococcus jannaschii (MJ1044)

International Nuclear Information System (INIS)

Rangarajan, Sarani; Jeyakanthan, Jeyaraman; Mridula, Palappetty; Sakamoto, Keiko; Kitamura, Yoshiaki; Agari, Yoshihiro; Shinkai, Akeo; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

2008-01-01

The ribosomal protein (L30e) from M. jannaschii was cloned from the gene MJ1044, expressed, purified and crystallized. The crystal belongs to the primitive tetragonal space group P4 3 and diffracted to 1.9 Å resolution. In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 × 0.20 × 0.35 mm) was obtained that belonged to the primitive tetragonal space group P4 3 , with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 Å, and diffracted to a resolution of 1.9 Å. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V M ) of 2.16 Å 3 Da −1

Structure determination of an integral membrane protein at room temperature from crystals in situ

International Nuclear Information System (INIS)

Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

2015-01-01

The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

Structure determination of an integral membrane protein at room temperature from crystals in situ

Energy Technology Data Exchange (ETDEWEB)

Axford, Danny [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Foadi, James [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Hu, Nien-Jen; Choudhury, Hassanul Ghani [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Iwata, So [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Kyoto University, Kyoto 606-8501 (Japan); Beis, Konstantinos [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Alguel, Yilmaz, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom)

2015-05-14

The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

International Nuclear Information System (INIS)

Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

2008-01-01

The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution. Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6 4 22, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å 3 Da −1 , which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan

Preparation, crystallization and preliminary X-ray characterization of a conserved hypothetical protein XC1692 from Xanthomonas campestris

International Nuclear Information System (INIS)

Chin, Ko-Hsin; Huang, Zhao-Wei; Wei, Kun-Chou; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

2005-01-01

A conserved hypothetical protein XC1692 from X. campestris pv. campestris has been overexpressed in E. coli. The purified recombinant protein crystallized in a variety of forms and diffracted to a resolution of at least 1.45 Å. Xanthomonas campestris pv. campestris strain 17 is a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. Its genome contains approximately 4500 genes, one third of which have no known structure and/or function yet are highly conserved among several different bacterial genuses. One of these gene products is XC1692 protein, containing 141 amino acids. It was overexpressed in Escherichia coli, purified and crystallized in a variety of forms using the hanging-drop vapour-diffusion method. The crystals diffract to at least 1.45 Å resolution. They are hexagonal and belong to space group P6 3 , with unit-cell parameters a = b = 56.9, c = 71.0 Å. They contain one molecule per asymmetric unit

PHOTOCATALYTIC ACTIVITIES of Ag+ DOPED ZIF-8 and ZIF-L CRYSTALS

Directory of Open Access Journals (Sweden)

Berna Topuz

2016-09-01

Full Text Available Photocatalysis is expected to contribute to the solution of environmental problems such as water and air pollution in the near future. The design of photocatalysts with high electron-hole generation rates, high surface areas and high light absorption capacities is crucial in producing sustainable and cost-effective photocatalytic processes. Titania, zirconia, copper oxide, zinc oxide, iron oxide are widely used photocatalysts which have good light absorption capacities with moderate surface areas depending on the synthesis conditions. In the last decade metal organic frameworks (MOFs have been used in photocatalytic applications due to their very high surface areas up to 1000s of m2/g and adequate light absorption capacities. In this study zeolitic imidazolate framework (ZIF based MOF photocatalytsts were prepared and the effect of silver (Ag doping on the photocatalytic activity of ZIF-8 and ZIF-L crystals was investigated. Ag doped ZIF-8 and ZIF-L crystals were prepared and their activities in the photocatalytic removal of methylene blue (MB dye under UV irradiation were determined for the first time in the literature. Doped ZIF-8 and ZIF-L crystals showed better photocatalytic activities compared to the undoped crystals. 100% of MB was removed with 5 mole% Ag+ doped ZIF-8 in 40 min. The photocatalytic activity decreased beyond 5% doping level since Ag+ ions may have segregated due to a possible solid state solubility limit of Ag+ ions in the crystal lattice of ZIF-8. ZIF-L crystals possessed lower photocatalytic activities compared to ZIF-8 crystals.

Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from bacillus thuringiensis: effect on insecticidal crystal proteins.

Science.gov (United States)

Tan, Y; Donovan, W P

2001-11-17

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.

Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi

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N. Leulliot

2013-01-01

Full Text Available Pyrococcus abyssi virus 1 (PAV1 was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23 that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137 at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer

International Nuclear Information System (INIS)

Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

2010-01-01

The RNA-binding protein Hfq from B. subtilis was crystallized using the hanging-drop vapour-diffusion method in two crystal forms that belonged to space groups I422 and F222; diffraction data were collected to 2.2 Å resolution from both forms. The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq–RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq–RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 Å, while the type 2 Hfq–RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 Å. Diffraction data were collected to a resolution of 2.20 Å from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis

Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

Directory of Open Access Journals (Sweden)

Chen Wang

Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

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

Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

Energy Technology Data Exchange (ETDEWEB)

Yamada, Mototsugu, E-mail: mototsugu-yamada@meiji.co.jp; Watanabe, Takashi; Baba, Nobuyoshi; Miyara, Takako; Saito, Jun; Takeuchi, Yasuo [Pharmaceutical Research Center, Meiji Seika Kaisha Ltd, 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567 (Japan)

2008-04-01

The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 86.39, c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent.

Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

International Nuclear Information System (INIS)

Yamada, Mototsugu; Watanabe, Takashi; Baba, Nobuyoshi; Miyara, Takako; Saito, Jun; Takeuchi, Yasuo

2008-01-01

The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4 3 2 1 2, with unit-cell parameters a = b = 86.39, c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent

Evaluation of Semi-supervised Learning for Classification of Protein Crystallization Imagery.

Science.gov (United States)

Sigdel, Madhav; Dinç, İmren; Dinç, Semih; Sigdel, Madhu S; Pusey, Marc L; Aygün, Ramazan S

2014-03-01

In this paper, we investigate the performance of two wrapper methods for semi-supervised learning algorithms for classification of protein crystallization images with limited labeled images. Firstly, we evaluate the performance of semi-supervised approach using self-training with naïve Bayesian (NB) and sequential minimum optimization (SMO) as the base classifiers. The confidence values returned by these classifiers are used to select high confident predictions to be used for self-training. Secondly, we analyze the performance of Yet Another Two Stage Idea (YATSI) semi-supervised learning using NB, SMO, multilayer perceptron (MLP), J48 and random forest (RF) classifiers. These results are compared with the basic supervised learning using the same training sets. We perform our experiments on a dataset consisting of 2250 protein crystallization images for different proportions of training and test data. Our results indicate that NB and SMO using both self-training and YATSI semi-supervised approaches improve accuracies with respect to supervised learning. On the other hand, MLP, J48 and RF perform better using basic supervised learning. Overall, random forest classifier yields the best accuracy with supervised learning for our dataset.

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

Science.gov (United States)

Lei, Li; Egli, Martin

2016-04-01

Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

Science.gov (United States)

Holmes, Anna M.; Monaco, Lisa; Barnes, Cindy; Spearing, Scott; Jenkins, Andy; Johnson, Todd; Mayer, Derek; Cole, Helen

2003-01-01

The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

Laboratory information management system for membrane protein structure initiative--from gene to crystal.

Science.gov (United States)

Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

2008-12-01

Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

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

Characterization of Baculovirus Insecticides Expressing Tailored Bacillus thuringiensis CryIA(b) Crystal Proteins

NARCIS (Netherlands)

Martens, John W M; Knoester, Marga; Weijts, Franci; Groffen, Sander J A; Hu, Zhihong; Bosch, Dirk; Vlak, Just M.

1995-01-01

Full-length, truncated, and mature forms of the CryIA(b) insecticidal crystal protein gene of Bacillus thuringiensis were engineered into the p10 locus of Autographa californica nuclear polyhedrosis virus (AcNPV). A signal sequence of Heliothis virescens juvenile hormone esterase was introduced at

Purification, crystallization and preliminary crystallographic analysis of GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

NARCIS (Netherlands)

Wu Hao,; Sun, L.; Brouns, S.J.J.; Fu, S.; Akerboom, A.P.; Li, X.; Oost, van der J.

2007-01-01

A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8

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.

Crystallization and preliminary X-ray characterization of a PaaX-like protein from Sulfolobus solfataricus P2

International Nuclear Information System (INIS)

Cao, Yi; Lou, Zhiyong; Sun, Yuna; Xue, Fei; Feng, Changzeng; Gong, Xiaocui; Yang, Dongmei; Bartlam, Mark; Meng, Zhaohui; Zhang, Keqin

2009-01-01

In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. PaaX is a global regulator of the phenylacetyl-coenzyme A catabolon that adjusts the expression of different operons to that of the paa-encoded central pathway. In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Diffraction data were obtained to a resolution of 3.0 Å using synchrotron radiation at the Photon Factory. The crystal belonged to space group P321, with unit-cell parameters a = 86.4, b = 86.4, c = 105.5 Å

Downstream Processability of Crystal Habit-Modified Active Pharmaceutical Ingredient

DEFF Research Database (Denmark)

Pudasaini, Nawin; Upadhyay, Pratik Pankaj; Parker, Christian Richard

2017-01-01

Efficient downstream processing of active pharmaceutical ingredients (APIs) can depend strongly on their particulate properties, such as size and shape distributions. Especially in drug products with high API content, needle-like crystal habit of an API may show compromised flowability and tablet......Efficient downstream processing of active pharmaceutical ingredients (APIs) can depend strongly on their particulate properties, such as size and shape distributions. Especially in drug products with high API content, needle-like crystal habit of an API may show compromised flowability...

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

Science.gov (United States)

Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

2015-10-08

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method.

Science.gov (United States)

Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

2013-11-01

Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method.

Crystal structure of Mycobacterium tuberculosis O6-methylguanine-DNA methyltransferase protein clusters assembled on to damaged DNA.

Science.gov (United States)

Miggiano, Riccardo; Perugino, Giuseppe; Ciaramella, Maria; Serpe, Mario; Rejman, Dominik; Páv, Ondřej; Pohl, Radek; Garavaglia, Silvia; Lahiri, Samarpita; Rizzi, Menico; Rossi, Franca

2016-01-15

Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair. © 2016 Authors; published by Portland Press Limited.

A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

Science.gov (United States)

Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

2014-01-01

Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

Preparation, crystallization and preliminary X-ray analysis of XC2382, an ApaG protein of unknown structure from Xanthomonas campestris

International Nuclear Information System (INIS)

Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

2005-01-01

A putative ApaG gene product from X. campestris pv. campestris was overexpressed in E. coli, purified and crystallized. The crystals diffracted to a resolution of at least 2.3 Å. Xanthomonas campestris pv. campestris is the causative agent of black rot, one of the major worldwide diseases of cruciferous crops. Its genome encodes approximately 4500 proteins, roughly one third of which have unknown function. XC2382 is one such protein, with a MW of 14.2 kDa. Based on a bioinformatics study, it was annotated as an ApaG gene product that serves multiple functions. The ApaG protein has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to a resolution of at least 2.30 Å. They are tetragonal and belong to space group P4 1/3 , with unit-cell parameters a = b = 57.6, c = 122.9 Å. There are two, three or four molecules in the asymmetric unit

Project in determination of crystal structure of nitrogen fixation proteins from azospirilum brasiliense and herbaspirilum seropedicae by synchrotron x-ray diffraction

International Nuclear Information System (INIS)

Barbosa, Valma M.; Leggs, Luciana A.; Delboni, Luis F.; Chubatsu, LedaS.; Souza, Emanuel M.; Machado, Hidevaldo B.; Yates, Geoffrey M.; Pedrosa, Fabio O.

1996-01-01

Full text. Biological nitrogen fixation is essential for maintaining the nitrogen cycle on earth and of high importance for Brazilian agriculture. The nitrogenase enzyme system, which provides the biochemical machinery for nitrogen fixation, consists of two component metalloproteins, the molybdenumiron (Mo Fe) protein and the iron (Fe) protein. Nitrogen fixation is a very energy-intensive process, requiring around 16 moles of ATP for each mol of N 2 fixed (reduced). As a consequence, synthesis and activity of nitrogenase is tighty regulated at two levels: general and specific. The general level regulation is mediated by the ntr (nitrogen regulation) system. Two gene products are involved: the ntrB gene product (NtrB) is responsible for the activation of the ntrC gene product (NtrC) by phosphorylating a conserved Asp54, which activates the expression of the nifA gene. The nif specific control system is mediated by the NifA protein, which binds to a DNA specific sequence (UAS, Upstream Activator Sequence) and activates nif promoter transcriptions by RNA polymerase- α54 , following ATP hydrolysis. The aim of this project is to solve the crystal structure of dinitrogenase reductase (iron protein) and dinitrogenase (molybdenum-iron protein) from Azospirilim brasiliense and the regulatory proteins NifA from Herbaspirillum seropedicae and NtrC Azospirillum brasiliense. The three dimensional structure of the proteins involved in this project will allow a better understanding of the mechanism of biological nitrogen fixation. To this end, the data collection will probably be done at the LNLS facilities which will be available in the near future. (author)

Project in determination of crystal structure of nitrogen fixation proteins from azospirilum brasiliense and herbaspirilum seropedicae by synchrotron x-ray diffraction

Energy Technology Data Exchange (ETDEWEB)

Barbosa, Valma M.; Leggs, Luciana A.; Delboni, Luis F.; Chubatsu, LedaS.; Souza, Emanuel M.; Machado, Hidevaldo B.; Yates, Geoffrey M.; Pedrosa, Fabio O. [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica

1996-12-31

Full text. Biological nitrogen fixation is essential for maintaining the nitrogen cycle on earth and of high importance for Brazilian agriculture. The nitrogenase enzyme system, which provides the biochemical machinery for nitrogen fixation, consists of two component metalloproteins, the molybdenumiron (Mo Fe) protein and the iron (Fe) protein. Nitrogen fixation is a very energy-intensive process, requiring around 16 moles of ATP for each mol of N{sub 2} fixed (reduced). As a consequence, synthesis and activity of nitrogenase is tighty regulated at two levels: general and specific. The general level regulation is mediated by the ntr (nitrogen regulation) system. Two gene products are involved: the ntrB gene product (NtrB) is responsible for the activation of the ntrC gene product (NtrC) by phosphorylating a conserved Asp54, which activates the expression of the nifA gene. The nif specific control system is mediated by the NifA protein, which binds to a DNA specific sequence (UAS, Upstream Activator Sequence) and activates nif promoter transcriptions by RNA polymerase-{sup {alpha}54}, following ATP hydrolysis. The aim of this project is to solve the crystal structure of dinitrogenase reductase (iron protein) and dinitrogenase (molybdenum-iron protein) from Azospirilim brasiliense and the regulatory proteins NifA from Herbaspirillum seropedicae and NtrC Azospirillum brasiliense. The three dimensional structure of the proteins involved in this project will allow a better understanding of the mechanism of biological nitrogen fixation. To this end, the data collection will probably be done at the LNLS facilities which will be available in the near future. (author)

Routine phasing of coiled-coil protein crystal structures with AMPLE

Directory of Open Access Journals (Sweden)

Jens M. H. Thomas

2015-03-01

Full Text Available Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing.

Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

Energy Technology Data Exchange (ETDEWEB)

Morand, Patrice [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France); Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble (France); Budayova-Spano, Monika [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France); Perrissin, Monique [Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble (France); Müller, Christoph W., E-mail: mueller@embl-grenoble.fr; Petosa, Carlo [European Molecular Biology Laboratory, Grenoble Outstation, BP 181, 38042 Grenoble CEDEX 9 (France)

2006-03-01

A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiation (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.

Active Photonic crystal fibers for high power applications

DEFF Research Database (Denmark)

Olausson, Christina Bjarnal Thulin

The photonic crystal ber technology provides means to realize bers optimized for high power operation, due to the large single-mode cores and the unique design exibility of the microstructure. The work presented in this thesis focuses on improving the properties of active photonic crystal bers...... contributed to the compounding of new and improved material compositions. The second part is an investigation of pump absorption in photonic crystal bers, demonstrating that the microstructure in photonic crystal bers improves the pump absorption by up to a factor of two compared to step-index bers....... This plays an important role in high power lasers and ampliers with respect to efficiency, packaging, and thermal handling. The third part of the work has involved developing tools for characterizing the mode quality and stability of large core bers. Stable, single-mode bers with larger cores are essential...

Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

Science.gov (United States)

Kent, Stephen Bh

2018-04-04

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

International Nuclear Information System (INIS)

Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

2013-01-01

Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners

Novel bacterial gas sensor proteins with transition metal-containing prosthetic groups as active sites.

Science.gov (United States)

Aono, Shigetoshi

2012-04-01

Gas molecules function as signaling molecules in many biological regulatory systems responsible for transcription, chemotaxis, and other complex physiological processes. Gas sensor proteins play a crucial role in regulating such biological systems in response to gas molecules. New sensor proteins that sense oxygen or nitric oxide have recently been found, and they have been characterized by X-ray crystallographic and/or spectroscopic analysis. It has become clear that the interaction between a prosthetic group and gas molecules triggers dynamic structural changes in the protein backbone when a gas sensor protein senses gas molecules. Gas sensor proteins employ novel mechanisms to trigger conformational changes in the presence of a gas. In gas sensor proteins that have iron-sulfur clusters as active sites, the iron-sulfur clusters undergo structural changes, which trigger a conformational change. Heme-based gas sensor proteins reconstruct hydrogen-bonding networks around the heme and heme-bound ligand. Gas sensor proteins have two functional states, on and off, which are active and inactive, respectively, for subsequent signal transduction in response to their physiological effector molecules. To fully understand the structure-function relationships of gas sensor proteins, it is vital to perform X-ray crystal structure analyses of full-length proteins in both the on and off states.

Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

Science.gov (United States)

Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

2015-06-01

Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RNA Crystallization

Science.gov (United States)

Golden, Barbara L.; Kundrot, Craig E.

2003-01-01

RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

Energy Technology Data Exchange (ETDEWEB)

Broecker, Jana; Klingel, Viviane; Ou, Wei-Lin; Balo, Aidin R.; Kissick, David J.; Ogata, Craig M.; Kuo, Anling; Ernst, Oliver P.

2016-10-12

In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometerbased X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.

Crystallization of protein–ligand complexes

International Nuclear Information System (INIS)

Hassell, Anne M.; An, Gang; Bledsoe, Randy K.; Bynum, Jane M.; Carter, H. Luke III; Deng, Su-Jun J.; Gampe, Robert T.; Grisard, Tamara E.; Madauss, Kevin P.; Nolte, Robert T.; Rocque, Warren J.; Wang, Liping; Weaver, Kurt L.; Williams, Shawn P.; Wisely, G. Bruce; Xu, Robert; Shewchuk, Lisa M.

2007-01-01

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals. Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks

Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD)

International Nuclear Information System (INIS)

Pandey, Nishant Kumar; Pal, Ravi Kant; Kashyap, Maruthi; Bhavesh, Neel Sarovar

2012-01-01

The Escherichia coli PapD-like protein (EcpD), from uropathogenic Escherichia coli (UPEC), which is a periplasmic chaperon of Yad fimbriae was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 1.67 Å resolution and belonged to space group C222 1 . Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Å resolution and belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å 3 Da −1 , with 59% solvent content. Initial phases were determined by molecular replacement

Fluorogen-activating proteins: beyond classical fluorescent proteins

Directory of Open Access Journals (Sweden)

Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Numerical study of slip system activity and crystal lattice rotation under wedge nanoindents in tungsten single crystals

Science.gov (United States)

Volz, T.; Schwaiger, R.; Wang, J.; Weygand, S. M.

2018-05-01

Tungsten is a promising material for plasma facing components in future nuclear fusion reactors. In the present work, we numerically investigate the deformation behavior of unirradiated tungsten (a body-centered cubic (bcc) single crystal) underneath nanoindents. A finite element (FE) model is presented to simulate wedge indentation. Crystal plasticity finite element (CPFE) simulations were performed for face-centered and body-centered single crystals accounting for the slip system family {110} in the bcc crystal system and the {111} slip family in the fcc system. The 90° wedge indenter was aligned parallel to the [1 ¯01 ]-direction and indented the crystal in the [0 1 ¯0 ]-direction up to a maximum indentation depth of 2 µm. In both, the fcc and bcc single crystals, the activity of slip systems was investigated and compared. Good agreement with the results from former investigations on fcc single crystals was observed. Furthermore, the in-plane lattice rotation in the material underneath an indent was determined and compared for the fcc and bcc single crystals.

Nonlinear Gain Saturation in Active Slow Light Photonic Crystal Waveguides

DEFF Research Database (Denmark)

Chen, Yaohui; Mørk, Jesper

2013-01-01

We present a quantitative three-dimensional analysis of slow-light enhanced traveling wave amplification in an active semiconductor photonic crystal waveguides. The impact of slow-light propagation on the nonlinear gain saturation of the device is investigated.......We present a quantitative three-dimensional analysis of slow-light enhanced traveling wave amplification in an active semiconductor photonic crystal waveguides. The impact of slow-light propagation on the nonlinear gain saturation of the device is investigated....

A Proposed Model for Protein Crystal Nucleation and Growth

Science.gov (United States)

Pusey, Marc; Curreri, Peter A. (Technical Monitor)

2002-01-01

How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme, based upon fluorescence, light, neutron, and X-ray scattering data, size exclusion chromatography experiments, dialysis kinetics, AFM, and modeling of growth rate data, from this and other laboratories. The first species formed is postulated to be a 'head to side' dimer. Through repeating associations involving the same intermolecular interactions this grows to a 4(sub 3) helix structure, that in turn serves as the basic unit for nucleation and subsequent crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates subsequent helix-helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub3)2(sub 1),2(sub 1) unit cell are filled. The process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the growth parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. In the general case for proteins the lack of a singularly defined

Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography.

Science.gov (United States)

Banigan, James R; Mandal, Kalyaneswar; Sawaya, Michael R; Thammavongsa, Vilasak; Hendrickx, Antoni P A; Schneewind, Olaf; Yeates, Todd O; Kent, Stephen B H

2010-10-01

The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

Science.gov (United States)

Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A; Kruse, Andrew C; Nurva, Shailika; Loland, Claus J; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H

2010-12-01

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

Science.gov (United States)

Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

2013-12-01

One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

Identification and cloning of two insecticidal protein genes from ...

African Journals Online (AJOL)

Bacillus thuringiensis (Bt) is the most widely applied type of microbial pesticide due to its high specificity and environmental safety. The activity of Bt is largely attributed to the insecticidal crystal protein encoded by the cry genes. Different insecticidal crystal proteins of Bt have different bioactivity against distinct agricultural ...

The charm of protein crystals--Structural biology at a glance in the International Year of Crystallography

International Nuclear Information System (INIS)

Su Xiaodong; Cao Qin

2014-01-01

Crystallography is a typical intellectual endeavor that has spanned human history for centuries. Through the persistent efforts of generations of scientists, crystallography has been transformed from a mathematical hypothesis to actual physical reality, mainly thanks to X-ray diffraction technology. 2014 is celebrated as the International Year of Crystallography (IYCr-2014), to commemorate that about 100 years ago, when Max von Laue in Germany and the father-and-son Braggs (William Henry Bragg and William Lawrence Bragg) in England pioneered the use of X-rays to determine the atomic structure of crystals; for this pioneering work they were awarded Nobel prizes for physics in the years of 1914 and 1915. This article is dedicated to the IYCr to describe the use of protein crystals, an application that has developed into protein crystallography and subsequently structural biology. In our overview of the history and future prospects of this field, we discuss in detail one example of caspase-6, to demonstrate how protein crystallography can help us understand the structure-function relationship of important proteins. (authors)

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the α-crystallin family

Energy Technology Data Exchange (ETDEWEB)

Hilario, Eduardo; Teixeira, Elaine Cristina; Pedroso, Gisele Audrei; Bertolini, Maria Célia [Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil); Medrano, Francisco Javier, E-mail: fjmedrano@yahoo.com [Departamento de Cristalografia de Proteínas, Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Caixa Postal 6192, CEP 13084-971, Campinas-SP (Brazil); Departamento de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara-SP (Brazil)

2006-05-01

XAC1151, a small heat-shock protein from X. axonopodis pv. citri belonging to the α-crystallin family, was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 Å. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

The purification, crystallization and preliminary structural characterization of human MAWDBP, a member of the phenazine biosynthesis-like protein family

International Nuclear Information System (INIS)

Herde, Petra; Blankenfeldt, Wulf

2006-01-01

The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution

The purification, crystallization and preliminary structural characterization of human MAWDBP, a member of the phenazine biosynthesis-like protein family

Energy Technology Data Exchange (ETDEWEB)

Herde, Petra; Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

2006-06-01

The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution.

Microscopic origins of anisotropic active stress in motor-driven nematic liquid crystals.

Science.gov (United States)

Blackwell, Robert; Sweezy-Schindler, Oliver; Baldwin, Christopher; Hough, Loren E; Glaser, Matthew A; Betterton, M D

2016-03-14

The cytoskeleton, despite comprising relatively few building blocks, drives an impressive variety of cellular phenomena ranging from cell division to motility. These building blocks include filaments, motor proteins, and static crosslinkers. Outside of cells, these same components can form novel materials exhibiting active flows and nonequilibrium contraction or extension. While dipolar extensile or contractile active stresses are common in nematic motor-filament systems, their microscopic origin remains unclear. Here we study a minimal physical model of filaments, crosslinking motors, and static crosslinkers to dissect the microscopic mechanisms of stress generation in a two-dimensional system of orientationally aligned rods. We demonstrate the essential role of filament steric interactions which have not previously been considered to significantly contribute to active stresses. With this insight, we are able to tune contractile or extensile behavior through the control of motor-driven filament sliding and crosslinking. This work provides a roadmap for engineering stresses in active liquid crystals. The mechanisms we study may help explain why flowing nematic motor-filament mixtures are extensile while gelled systems are contractile.

Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

Energy Technology Data Exchange (ETDEWEB)

Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan [Institute of Biotechnology CAS, Videnska 1083, 142 20 Prague (Czech Republic)

2015-10-27

The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.

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

Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamer.

Science.gov (United States)

Baba, Seiki; Someya, Tatsuhiko; Kawai, Gota; Nakamura, Kouji; Kumasaka, Takashi

2010-05-01

The Hfq protein is a hexameric RNA-binding protein which regulates gene expression by binding to RNA under the influence of diverse environmental stresses. Its ring structure binds various types of RNA, including mRNA and sRNA. RNA-bound structures of Hfq from Escherichia coli and Staphylococcus aureus have been revealed to have poly(A) RNA at the distal site and U-rich RNA at the proximal site, respectively. Here, crystals of a complex of the Bacillus subtilis Hfq protein with an A/G-repeat 7-mer RNA (Hfq-RNA) that were prepared using the hanging-drop vapour-diffusion technique are reported. The type 1 Hfq-RNA crystals belonged to space group I422, with unit-cell parameters a = b = 123.70, c = 119.13 A, while the type 2 Hfq-RNA crystals belonged to space group F222, with unit-cell parameters a = 91.92, b = 92.50, c = 114.92 A. Diffraction data were collected to a resolution of 2.20 A from both crystal forms. The hexameric structure of the Hfq protein was clearly shown by self-rotation analysis.

Identification of a macromolecular crystal growth inhibitor in human urine as osteopontin

DEFF Research Database (Denmark)

Sørensen, Steen; Justesen, S J; Johnsen, A H

1995-01-01

, an unidentified protein rich in uronic acid, and uropontin have all been described as possessing such activity. We have recently isolated an unknown inhibitor of calcium oxalate crystal growth that co-eluted with trypsin inhibitor in several separation steps, which suggested its identity. The aim of the present......Macromolecules occurring in human urine inhibit the growth and/or aggregation of calcium oxalate crystals and may prevent the formation of kidney stones. Attention has focused particularly on proteins, as these seem to be most responsible for the inhibitory activity; three proteins, nephrocalcin...... study was to outline a simple procedure for isolating and identifying this inhibitor. Purification was done as follows: precipitation of the major proteins (albumin and uromucoid) with trichloroacetic acid, followed by anion exchange chromatography, hydroxyapatite chromatography, anion exchange...

Crystal structure of the extracellular domain of human myelin protein zero

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C. (WSU-MED); (NWU)

2012-03-27

different mutations in the MPZ gene leading to peripheral neuropathy in patients have been reported worldwide (http://www.molgen. ua.ac.be/CMTMutations). All identified mutations resulting in a change or deletion of amino acid residues in MPZ give rise to neuropathy with the exception of R215L, which instead causes a benign polymorphism. Furthermore, more detailed analysis has classified the MPZ mutations into two major groups. In the first group, the mutations disrupt the intracellular processing of MPZ and are primarily associated with early onset neuropathy. It has been proposed that the mutated MPZ is trapped inside the cell rather than being transported to the plasma membrane. However, other evidence suggests that the mutated MPZ protein is expressed on the plasma membrane, but dominant-negatively disrupts the structure of myelin. In the second group, the MPZ mutations are associated with late onset neuropathy as these mutations cause only mild demyelination. The underlying mechanism is elusive with the hypothesis being that the second group of mutations cause minor abnormalities in the myelin sheath that over time may lead to aberrant Schwann cell-axon interactions and subsequently to axonal degeneration. The crystal structure of the extracellular domain of human MPZ (hP0ex) fused with maltose binding protein (MBP) is reported at 2.1 {angstrom} resolution. While the crystal structure of rat MPZ extracellular domain (rP0ex) is available, the crystal structure of the human counterpart is useful for the analysis of the two homologs as well as a comparison between the two species. The hP0ex molecule reveals subtle structural variations between two homologs allowing comparison of the human myelin protein zero to that of the rat protein. The alignment of these homologs is shown in Figure 1(a).

Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

Energy Technology Data Exchange (ETDEWEB)

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

2004-12-17

Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

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

Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

International Nuclear Information System (INIS)

Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

2007-01-01

A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2 1 and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2 1 , with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å 3 Da −1 and a solvent content of 50%

Powder diffraction from a continuous microjet of submicrometer protein crystals.

Science.gov (United States)

Shapiro, D A; Chapman, H N; Deponte, D; Doak, R B; Fromme, P; Hembree, G; Hunter, M; Marchesini, S; Schmidt, K; Spence, J; Starodub, D; Weierstall, U

2008-11-01

Atomic-resolution structures from small proteins have recently been determined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865-11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules.