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X-ray Diffraction Data Set (x-ray + diffraction_data_set)
Kinds of X-ray Diffraction Data Set Selected AbstractsCyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2009Soheila Emamzadah 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. [source] Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholinoethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3,Å spacing has been collected. [source] Purification, crystallization and X-ray diffraction analysis of the extracellular part of the human Fc receptor for IgA, Fc,RI (CD89)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Katja Wenig Fc,RI is the predominant receptor for IgA in the serum. Nevertheless, the interaction between the molecules that finally leads to an immune response is poorly understood. To investigate the structural requirements for IgA binding, the extracellular region of Fc,RI was cloned and overexpressed in Escherichia coli. The resulting inclusion-body protein was refolded and purified. Despite its deglycosylated state, this recombinant Fc,RI retained its ability to bind human IgA. The protein crystallized spontaneously as microcrystalline needles. Recrystallization yielded crystals belonging to a primitive monoclinic space group. A complete 2.8,Å resolution X-ray diffraction data set was collected using synchrotron radiation. [source] Crystallization and preliminary X-ray analysis of the complex of porcine pancreatic elastase and a hybrid squash inhibitorACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002Kai Hilpert A hybrid inhibitor consisting of the scaffold of a squash-type inhibitor and a specific inhibitory peptide optimized from the third domain of ovomucoid inhibitor from turkey against porcine pancreatic elastase was synthesized by peptide synthesis. The complex formed by this hybrid inhibitor and the porcine pancreatic elastase was crystallized using the hanging-drop method with citrate in the crystallization solution. The space group was determined to be P212121, with unit-cell parameters a = 56.33, b = 56.44, c = 72.76,Å. A complete X-ray diffraction data set was collected under cryogenic conditions to 1.8,Å. [source] Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Francieli Colussi The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source. [source] Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Qianda Lu 5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA of Bacillus subtilis and the protein was overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH, tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7,Å. [source] Crystallization and preliminary X-ray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Weijie You Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts ,-galactose to ,-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283,K. An X-ray diffraction data set was collected to a resolution of 1.9,Å from a single flash-cooled crystal that belonged to space group P212121. [source] Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Lei Jin NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 118.7, c = 106.0,Å. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2,Å resolution and a clear molecular-replacement solution was obtained for solution of the structure. [source] Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Trypanosoma brucei gambiense glycerol kinaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Emmanuel Oluwadare Balogun In the bloodstream forms of human trypanosomes, glycerol kinase (GK; EC 2.7.1.30) is one of the nine glycosomally compartmentalized enzymes that are essential for energy metabolism. In this study, a recombinant Trypanosoma brucei gambiense GK (rTbgGK) with an N-terminal cleavable His6 tag was overexpressed, purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. A complete X-ray diffraction data set to 2.75,Å resolution indicated that the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 63.84, b = 121.50, c = 154.59,Å. The presence of two rTbgGK molecules in the asymmetric unit gives a Matthews coefficient (VM) of 2.5,Å3,Da,1, corresponding to 50% solvent content. [source] Use of thallium to identify monovalent cation binding sites in GroELACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Philip D. Kiser GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D7 symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg2+ and K+, to bind and hydrolyze ATP. A K+ -binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K+ -binding site may exist within GroEL. Because K+ has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K+ mimetic Tl+ was incorporated into GroEL crystals, a moderately redundant 3.94,Å resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K+ next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl+ for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low. [source] Crystallization and preliminary crystallographic studies of the single-chain variable fragment of antibody chA21 in complex with an N-terminal fragment of ErbB2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Yang Liu ErbB2 is a transmembrane tyrosine kinase, the overexpression of which causes abnormality and disorder in cell signalling and leads to cell transformation. Previously, an anti-ErbB2 single-chain chimeric antibody chA21 that specifically inhibits the growth of ErbB2-overexpressing cancer cells in vitro and in vivo was developed. Here, an antibody,antigen complex consisting of the single-chain variable fragment (scFv) of chA21 and an N-terminal fragment (residues 1,192, named EP I) of the ErbB2 extracellular domain was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.45,Å resolution from a single flash-cooled crystal; the crystal belonged to space group P212121. [source] Crystallization and preliminary X-ray analysis of a rat aldose reductase-like protein (AKR1B14)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Roland Chung Mouse vas deferens protein/aldo-keto reductase 1B7 (AKR1B7) is involved in the detoxification of isocaproaldehyde, a steroidogenesis byproduct, and of 4-hydroxynonenal formed by lipid peroxidation. The rat orthologue of AKR1B7 has recently been named AKR1B14 in the AKR superfamily. Recombinant AKR1B14 was expressed in a bacterial system and purified to homogeneity. The purified protein was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method and an X-ray diffraction data set was collected to 1.86,Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 50.66, b = 69.14, c = 72.27,Å, , = 96.4°. This is the first crystallization report of a rodent AKR1B7 orthologue. [source] Crystallization and preliminary crystallographic analysis of the second RRM of Pub1 from Saccharomyces cerevisiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Yingji Cui mRNA stability is elaborately regulated by elements in the mRNA transcripts and their cognate RNA-binding proteins, which play important roles in regulating gene expression at the post-transcriptional level in eukaryotes. Poly(U)-binding protein 1 (Pub1), which is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae, is involved in the regulation of mRNA turnover as a trans -acting factor. It binds to transcripts containing the AU-rich element in order to protect them from degradation. Pub1 contains three RNA-recognition motifs (RRMs) which play significant roles in mRNA binding at AU-rich elements and stabilizer elements. In this study, the second RRM of Pub1 was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283,K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group H3. [source] Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A2 with low catalytic activity from Bothrops jararacussu venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2006L. C. Corrêa For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s. [source] Crystallization of recombinant Haemophilus influenzaee (P4) acid phosphataseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2006Zhonghui Ou Haemophilus influenzae infects the upper respiratory tract of humans and can cause infections of the middle ear, sinuses and bronchi. The virulence of the pathogen is thought to involve a group of surface-localized macromolecular components that mediate interactions at the host,pathogen interface. One of these components is lipoprotein e (P4), which is a class C acid phosphatase and a potential vaccine candidate for nontypeable H. influenzae infections. This paper reports the crystallization of recombinant e (P4) and the acquisition of a 1.7,Å resolution native X-ray diffraction data set. The space group is P42212, with unit-cell parameters a = 65.6, c = 101.4,Å, one protein molecule per asymmetric unit and 37% solvent content. This is the first report of the crystallization of a class C acid phosphatase. [source] Weak interactions in chain polymers [M(,- X)2L2], (M = Zn, Cd; X = Cl, Br; L = substituted pyridine) , an electron density studyACTA CRYSTALLOGRAPHICA SECTION B, Issue 5 2009Ruimin Wang The experimental electron-density distributions in crystals of five chain polymers [M(,- X)2(py)2] (M = Zn, Cd; X = Cl, Br; py = 3,5-substituted pyridine) have been obtained from high-resolution X-ray diffraction data sets (sin,,/, > 1.1,Å,1) at 100,K. Topological analyses following Bader's `Atoms in Molecules' approach not only confirmed the existence of (3, ,1) critical points for the chemically reasonable and presumably strong covalent and coordinative bonds, but also for four different secondary interactions which are expected to play a role in stabilizing the polymeric structures which are unusual for Zn as the metal center. These weaker contacts comprise intra- and inter-strand C,H...X,M hydrogen bonds on the one hand and C,X...X,C interhalogen contacts on the other hand. According to the experimental electron-density studies, the non-classical hydrogen bonds are associated with higher electron density in the (3, ,1) critical points than the halogen bonds and hence are the dominant interactions both with respect to intra- and inter-chain contacts. [source] Charge-density study on cyclosporine AACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009S. K. J. Johnas Two single-crystal X-ray diffraction data sets of cyclosporine A were measured to high resolution using synchrotron radiation at temperatures of 5 and 90,K. They allowed an accurate determination of its molecular and electronic structure. Three electron-density models based on pseudoatom scattering factors were compared in terms of derived bond topological properties and in terms of electron-density differences on a grid. In one model multipole parameters were freely refined, whereas in the other two models the density was built up from fixed database parameters from the invariom database and University at Buffalo Databank. The data quality not only allowed benchmarking of the quality of both databases with the refined density, but also judgement of the feasibility of a multipole refinement of a larger oligopeptide structure such as cyclosporine A. Both databases performed equally well and reproduced the experimentally determined charge density satisfactorily. [source] Crystallization and preliminary X-ray crystallographic analysis of the [NiFe]-hydrogenase maturation factor HypF1 from Ralstonia eutropha H16ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Gordon Winter The hydrogenase maturation factor HypF1 is a truncated but functional version of the HypF protein. HypF is known to be involved in the supply of the CN, ligands of the active site of [NiFe]-hydrogenases, utilizing carbamoyl phosphate as a substrate. The first crystallization and preliminary X-ray studies of HypF1 from Ralstonia eutropha H16 are reported here. Crystals of HypF1 (394 amino acids, 40.7,kDa) were obtained by the sitting-drop vapour-diffusion technique using sodium formate as a precipitant. The crystals belonged to space group I222, with unit-cell parameters a = 79.7, b = 91.6, c = 107.2,Å. Complete X-ray diffraction data sets were collected at 100,K from native crystals and from a platinum derivative to a maximum resolution of 1.65,Å. [source] Crystallization and preliminary crystallographic studies of the catalytic subunits of human pyruvate dehydrogenase phosphatase isoforms 1 and 2ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Junko Kato Pyruvate dehydrogenase phosphatase (PDP) is a mitochondrial serine phosphatase that activates phosphorylated pyruvate dehydrogenase complex by dephosphorylation. In humans, two PDP isoforms (1 and 2) have been identified. PDP1 is composed of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r), whereas PDP2 consists of only a catalytic subunit (PDP2c). Both PDP1c and PDP2c have been crystallized individually and complete X-ray diffraction data sets have been collected to 2.45 and 2.0,Å resolution, respectively. The PDP1c crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 65.1, c = 216.1,Å. The asymmetric unit is expected to contain one molecule, with a Matthews coefficient VM of 2.56,Å3,Da,1. The PDP2c crystals belonged to space group P212121, with unit-cell parameters a = 53.6, b = 69.1, c = 109.7,Å. The asymmetric unit is expected to contain one molecule, with a Matthews coefficient VM of 1.91,Å3,Da,1. [source] Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas udaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Annelies Van Hoorebeke Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [,- d -glucopyranosyl-(1,4)- d -glucopyranose] to ,- d -glucose-1-phosphate and d -glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity. [source] Crystallization and preliminary X-ray crystallographic analysis of Thermus thermophilus transcription elongation complex bound to Gfh1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Shunsuke Tagami RNA polymerase (RNAP) elongates RNA by iterative nucleotide-addition cycles (NAC). A specific structural state (or states) of RNAP may be the target of transcription elongation factors. Gfh1, a Thermus thermophilus Gre-family protein, inhibits NAC. To elucidate which RNAP structural state Gfh1 associates with, the T. thermophilus RNAP elongation complex (EC) was cocrystallized with Gfh1. Of the 70 DNA/RNA scaffolds tested, two (for EC1 and EC2) were successfully crystallized. In the presence of Gfh1, EC1 and EC2 yielded crystals belonging to space group P21 with similar unit-cell parameters (crystals 1 and 2, respectively). X-ray diffraction data sets were obtained at 3.6 and 3.8,Å resolution, respectively. [source] Purification, crystallization and preliminary X-ray analysis of the PCNA2,PCNA3 complex from Sulfolobus tokodaii strain 7ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Akito Kawai Crenarchaeal PCNA is known to consist of three subunits (PCNA1, PCNA2 and PCNA3) that form a heterotrimer (PCNA123). Recently, another heterotrimeric PCNA composed of only PCNA2 and PCNA3 was identified in Sulfolobus tokodaii strain 7 (stoPCNAs). In this study, the purified stoPCNA2,stoPCNA3 complex was crystallized by hanging-drop vapour diffusion. The crystals obtained belonged to the orthorhombic space groups I222 and P21212, with unit-cell parameters a = 91.1, b = 111.8, c = 170.9,Å and a = 91.1, b = 160.6, c = 116.6,Å, respectively. X-ray diffraction data sets were collected to 2.90,Å resolution for the I222 crystals and to 2.80,Å resolution for the P21212 crystals. [source] Purification, crystallization and preliminary X-ray crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Xinyuan Duan Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21,Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected to resolutions of 2.3 and 3.9,Å from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C2221, with unit-cell parameters a = 47.465, b = 97.574, c = 249.215,Å for the native crystals. [source] Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteriaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Gui-Lan Li All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. Two dUTPase-encoding genes, yncF and yosS, have been identified in Bacillus subtilis. The gene dut, encoding dUTPase from the dental pathogen Streptococcus mutans, was amplified from S. mutans genomic DNA. The three genes were cloned into expression vectors and overexpressed at high levels in Escherichia coli. Each protein was purified in two steps using chromatographic methods. Crystals of the YosS and YncF proteins and of S. mutans dUTPase were obtained using the vapour-diffusion method. X-ray diffraction data sets were collected from crystals of selenomethionine-labelled YosS and S. mutans dUTPase to resolutions of 2.3 and 1.7,Å, respectively. The crystal of native YncF diffracted to 2.7,Å resolution. [source] | ||||||||||