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Molecular-replacement Solution (molecular-replacement + solution)
Selected AbstractsCrystallization and preliminary analysis of a water-forming NADH oxidase from Lactobacillus sanfranciscensisACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2004George T. Lountos Single crystals have been obtained of NADH oxidase (Nox), a flavoenzyme cloned from Lactobacillus sanfranciscensis. The enzyme catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water, without releasing hydrogen peroxide after the reduction of the first equivalent of NAD(P)H. The enzyme crystallizes in space group P212121, with unit-cell parameters a = 59.6, b = 92.6, c = 163.5,Å. The crystals diffract to 1.85,Å resolution using synchrotron radiation. Matthews coefficient calculations suggest the presence of two molecules per asymmetric unit (VM = 2.3,Å3,Da,1, 45.5% solvent content), which has been confirmed by the molecular-replacement solution using a search molecule derived from NADH peroxidase (PDB code 1f8w). [source] Crystallization and preliminary X-ray diffraction studies of a fungal hydrolase from Ophiostoma novo-ulmiACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2004Michail N. Isupov Dutch elm disease fungus Ophiostoma novo-ulmi contains a hydrolase activity which catalyses the resolution of racemic ethyl naproxen to the corresponding acid. The recombinant enzyme has been crystallized by the vapour-diffusion method in two crystal forms. The crystals of the first form belong to space group P21212, with unit-cell parameters a = 115.9, b = 174.4, c = 62.1,Å. The enzyme also crystallizes in space group P21212, with unit-cell parameters a = 72.9, b = 212.7, c = 61.7,Å. Synchrotron data have been collected for both crystal forms to 2.6 and 2.3,Å, respectively. A molecular-replacement solution has been found using a remote starting model of a bacterial esterase (23% sequence identity) for both crystal forms. Multicrystal averaging has resulted in interpretable electron-density maps. [source] Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigenACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Shuh-Chyung Song The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293,K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4,8,mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25,Å resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75,Å resolution. Data were collected that indicated that the crystals belonged to space group P212121, with unit-cell parameters a = 57.9, b = 77.1, c = 118.1,Å and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP). [source] Expression, purification, crystallization and preliminary X-ray crystallographic studies of a psychrophilic cellulase from Pseudoalteromonas haloplanktisACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Sébastien Violot The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase. To date, a three-dimensional structure of a psychrophilic cellulase has been lacking. Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures. The catalytic core domain of the psychrophilic cellulase CelG from P. haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8,Å has been collected. The space group was found to be P212121, with unit-cell parameters a = 135.1, b = 78.4, c = 44.1,Å. A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found. [source] Crystallization, preliminary X-ray analysis and molecular-replacement solution of haemoglobin-II from the fish matrinxã (Brycon cephalus)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2003J. C. L. Fonseca Haemoglobins constitute a set of proteins with interesting structural and functional properties, especially when the two large animal groups reptiles and fishes are focused on. Here, the crystallization and preliminary X-ray analysis of haemoglobin-II from the South American fish matrinxã (Brycon cephalus) is reported. X-ray diffraction data have been collected to 3.0,Å resolution using synchrotron radiation (LNLS). Crystals were determined to belong to space group P21 and preliminary structural analysis revealed the presence of two tetramers in the asymmetric unit. The structure was determined using the standard molecular-replacement technique. [source] Structure of cytochrome c2 from Rhodospirillum centenumACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001A. Camara-Artigas Cytochrome c2 from the purple photosynthetic bacterium Rhodospirillum centenum has been crystallized by the sitting-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 29.7, b = 59.9, c = 65.4,Å, and diffract to a resolution limit of 1.7,Å. The Fe-atom position was determined from its anomalous scattering contribution and a molecular-replacement solution was calculated. The correctness of the solution was confirmed by parallel isomorphous replacement studies. The resulting model has a type I cytochrome fold with two features, an extended ,-helix and a surface-charge distribution, that are distinctive to this protein. The implications of these structural features for the ability of the cytochrome to serve as an electron carrier are discussed. [source] Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001Erica Ollmann Saphire An intact human immunoglobulin with a full-length hinge has been crystallized for the first time in a form in which all of the Ig domains are ordered. The IgG1 antibody b12 is one of only three known monoclonal antibodies described that potently neutralize a broad range of HIV-1 primary isolates. It binds to an epitope overlapping the conserved CD4 binding site on the viral surface antigen gp120. Hexagonal crystals corresponding to space group R32 were grown from 0.8,M ammonium sulfate, with unit-cell parameters a = b = 271.3, c = 175.2,Å and one molecule per asymmetric unit. The crystals diffract to 2.8,Å and a preliminary molecular-replacement solution indicates that all 12 Ig domains of the antibody can be resolved. [source] A systematic case study on using NMR models for molecular replacement: p53 tetramerization domain revisitedACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000Yu Wai Chen Molecular replacement using search models derived from nuclear magnetic resonance (NMR) spectroscopy has often proved problematic. It has been known for some time that the overall differences in atomic positions (r.m.s.d.) between the crystalline and the solution states of the same protein are of the order of 1,2,Å and approach the limit of molecular replacement. In most cases, this structural difference is a result of calculating the NMR structure with insufficient data, yielding an NMR structure of limited accuracy. A systematic case study was performed to investigate the use of NMR models for molecular replacement on the p53 tetramerization domain: NMR search models of varying degrees of accuracy were employed to solve phases for the 1.5,Å X-ray diffraction data. An approximate correlation was found between the accuracy of the NMR search model and the clarity and quality of the molecular-replacement solution. It was found that ensemble models perform better than single averaged models and have a larger tolerance in model inaccuracy. Also, distance-derived B factors can improve the performance of single models. [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] Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Zhijie Li Acyl-CoA dehydrogenase [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyzes the first reaction step in mitochondrial fatty-acid ,-oxidation. Here, the very-long-chain acyl-CoA dehydrogenase from Caenorhabditis elegans (cVLCAD) has been cloned and overexpressed in Escherichia coli strain BL21 (DE3). Interestingly, unlike other very-long-chain acyl-CoA dehydrogenases, cVLCAD was found to form a tetramer by size-exclusion chromatography coupled with in-line static light-scattering, refractive-index and ultraviolet measurements. Purified cVLCAD (12,mg,ml,1) was successfully crystallized by the hanging-drop vapour-diffusion method under conditions containing 100,mM Tris,HCl pH 8.0, 150,mM sodium chloride, 200,mM magnesium formate and 13% PEG 3350. The crystal has a tetragonal form and a complete diffraction data set was collected and processed to 1.8,Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 138.6, b = 116.7, c = 115.3,Å, , = , = 90.0, , = 124.0°. A self-rotation function indicated the existence of one noncrystallographic twofold axis. A preliminary molecular-replacement solution further confirmed the presence of two molecules in one asymmetric unit, which yields a Matthews coefficient VM of 2.76,Å3,Da,1 and a solvent content of 55%. [source] Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Miranda L. Byrne-Steele Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3,kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5,Å. A single MbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40,Å resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained. [source] Crystallization and preliminary X-ray crystallographic characterization of a public CMV-specific TCR in complex with its cognate antigenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Jean-Baptiste Reiser The T-cell response to human cytomegalovirus is characterized by a dramatic reduction of clonal diversity in patients undergoing chronic inflammation or immunodepression. In order to check whether all the selected high-avidity T-cell clones recognize the immunodominant pp65 peptide antigen pp65495,503 (NLVPMVATV) presented by the major histocompatibility complex (MHC) molecule HLA-A2 in a similar manner, several public high-affinity T-cell receptors (TCRs) specific for the pp65495,503,HLA-A2 complex have been investigated. Expression, purification and crystallization were performed and preliminary crystallographic data were collected to 4.7,Å resolution for the RA15 TCR in complex with the pp65495,503,HLA-A2 complex. Comparison of the RA15,pp65495,503,HLA-A2 complex molecular-replacement solution with the structure of another high-affinity pp65495,503,HLA-A2-specific TCR, RA14, shows a shared docking mode, indicating that the clonal focusing could be accompanied by the selection of a most favoured peptide-readout mode. However, the position of the RA15 V, domain is significantly shifted, suggesting a different interatomic interaction network. [source] Crystallization and preliminary X-ray diffraction analysis of crotoxin B from Crotalus durissus collilineatus venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009G. H. M. Salvador Crotoxin B is a basic phospholipase A2 found in the venom of several Crotalus durissus ssp. rattlesnakes and is one of the subunits that constitute crotoxin, the main component of the venom of these snakes. This heterodimeric toxin is related to important envenomation effects such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, the first structural data only became available in 2007 (for crotoxin B from C. durissus terrificus) and showed an ambiguous result for the biological assembly, which could be either dimeric or tetrameric. In this work, the crystallization, X-ray diffraction data collection at 2.2,Å resolution and molecular-replacement solution of a dimeric complex formed by two crotoxin B isoforms from C. durissus collilineatus venom is presented. [source] Cloning, purification, crystallization and preliminary X-ray analysis of the receiver domain of the histidine kinase CKI1 from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Klumpler The receiver domain (RD) of a sensor histidine kinase (HK) catalyses the transphosphorylation reaction during the action of HKs in hormonal and abiotic signalling in plants. Crystals of the recombinant RD of the Arabidopsis thaliana HK CYTOKININ-INDEPENDENT1 (CKI1RD) have been obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant and glycerol as a cryoprotectant. The crystals diffracted to approximately 2.4,Å resolution on beamline BW7B of the DORIS-III storage ring. The diffraction improved significantly after the use of a non-aqueous cryoprotectant. Crystals soaked in Paratone-N diffracted to at least 2.0,Å resolution on beamline BW7B and their mosaicity decreased more than tenfold. The crystals belonged to space group C2221, with unit-cell parameters a = 54.46, b = 99.82, c = 79.94,Å. Assuming the presence of one molecule of the protein in the asymmetric unit gives a Matthews coefficient VM of 2.33,Å3,Da,1. A molecular-replacement solution has been obtained and structure refinement is in progress. [source] Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragaceaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009A. E. Mechaly Sea anemones produce water-soluble toxins that have the ability to interact with cell membranes and form pores within them. The mechanism of pore formation is based on an initial binding step followed by oligomerization and membrane insertion. Although the final structure of the pore remains unclear, biochemical studies indicate that it consists of a tetramer with a functional radius of ,1.1,nm. Since four monomers seem to be insufficient to build a pore of this size, the currently accepted model suggests that lipids might also participate in its structure. In this work, the crystallization and preliminary crystallographic analysis of two crystal forms of fragaceatoxin C (FraC), a newly characterized actinoporin from Actinia fragacea, are described. The crystals diffracted up to 1.8,Å resolution and the preliminary molecular-replacement solution supports an oligomeric structure of about 120,Å in diameter. [source] Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolyticaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2007Chinthalapudi Krishna Entamoeba histolytica, the causative agent of human amoebiasis, is essentially anaerobic, requiring a small amount of oxygen for growth. It cannot tolerate the higher concentration of oxygen present in human tissues or blood. However, during tissue invasion it is exposed to a higher level of oxygen, leading to oxygen stress. Cysteine, which is a vital thiol in E. histolytica, plays an essential role in its oxygen-defence mechanisms. The major route of cysteine biosynthesis in this parasite is the condensation of O -acetylserine with sulfide by the de novo cysteine-biosynthetic pathway, which involves cysteine synthase (EhCS) as a key enzyme. In this study, EhCS was cloned, expressed in Escherichia coli and purified by affinity and size-exclusion chromatography. The purified protein was crystallized in space group P41 with two molecules per asymmetric unit and a complete data set was collected to a resolution of 1.86,Å. A molecular-replacement solution was obtained using the Salmonella typhimuriumO -acetylserine sulfhydrylase structure as a probe and had a correlation coefficient of 37.7% and an R factor of 48.8%. [source] Crystallization and preliminary crystallographic studies of the recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006Sergio Martínez-Rodríguez Dihydropyrimidinases are involved in the reductive pathway of pyrimidine degradation, catalysing the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N -carbamoyl ,-amino acids. This enzyme has often been referred to as hydantoinase owing to its industrial application in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins. Recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been expressed, purified and crystallized. Crystallization was performed using the counter-diffusion method with capillaries of 0.3,mm inner diameter. Crystals of SmelDhp suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to ensure mass transport controlled by diffusion. X-ray data were collected to a resolution of 1.85,Å. The crystal belongs to the orthorhombic space group C2221, with unit-cell parameters a = 124.89, b = 126.28, c = 196.10,Å and two molecules in the asymmetric unit. A molecular-replacement solution has been determined and refinement is in progress. [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] With phases: how two wrongs can sometimes make a rightACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Pietro Roversi In isolation, both weak isomorphous/anomalous difference signals from heavy-atom derivatization and phases from partial molecular-replacement solutions for a subset of the asymmetric unit often fall short of producing interpretable electron-density maps. Phases generated from very partial molecular-replacement models (if generated carefully) can be used to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust finding of the sites using Patterson interpretation or direct methods. Additional advantages are that using molecular-replacement phases to define the heavy-atom substructure avoids the need for subsequent hand determination and/or origin-choice reconciliation and that the partial model can be used to aid the mask determination during solvent flattening. Two case studies are presented in which it was only by combining experimental and molecular-replacement phasing approaches that the crystal structures could be determined. [source] Imperfect pseudo-merohedral twinning in crystals of fungal fatty acid synthaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2009Simon Jenni The recent high-resolution structures of fungal fatty acid synthase (FAS) have provided new insights into the principles of fatty acid biosynthesis by large multifunctional enzymes. The crystallographic phase problem for the 2.6,MDa fungal FAS was initially solved to 5,Å resolution using two crystal forms from Thermomyces lanuginosus. Monoclinic crystals in space group P21 were obtained from orthorhombic crystals in space group P212121 by dehydration. Here, it is shown how this space-group transition induced imperfect pseudo-merohedral twinning in the monoclinic crystal, giving rise to a Moiré pattern-like interference of the two twin-related reciprocal lattices. The strategy for processing the twinned diffraction images and obtaining a quantitative analysis is presented. The twinning is also related to the packing of the molecules in the two crystal forms, which was derived from self-rotation function analysis and molecular-replacement solutions using a low-resolution electron microscopy map as a search model. [source] | ||||||||||