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Vapour-diffusion Technique (vapour-diffusion + technique)
Kinds of Vapour-diffusion Technique Selected AbstractsAtomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnelsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010A. Stsiapanava The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P212121, while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22,Å, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent. [source] Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environmentACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Ekaterina Morgunova The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5,Å resolution to a final R factor of 23.7% (Rfree = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells. [source] Purification, crystallization and preliminary X-ray analysis of Triatoma virus (TrV) from Triatoma infestansACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Gabriela S. Rozas-Dennis Triatoma virus (TrV) is a viral pathogen of the blood-sucking reduviid bug Triatoma infestans, the most important vector of American human trypanosomiasis (Chagas' disease). TrV has been putatively classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work describes the purification of TrV particles from infected T. infestans and their crystallization and preliminary crystallographic analyses. Two different crystal forms, rhombohedral and orthorhombic, were obtained at room temperature by the hanging-drop vapour-diffusion technique using polyethylene glycol and polyethylene glycol monomethylether as precipitants. The rhombohedral crystals have unit-cell parameters a = b = 306.6, c = 788.4,Å (hexagonal setting), diffract to 3.2,Å resolution and contain one-third of the viral particle per asymmetric unit. The orthorhombic crystals have cell parameters a = 336, b = 351, c = 332,Å, diffract to about 2.5,Å resolution, and contain one-half of a virus particle in the asymmetric unit. A complete diffraction data set has been collected to 3.2,Å resolution, using synchrotron radiation, from a single rhombohedral crystal under cryogenic conditions. [source] Crystallization and preliminary crystallographic studies of the D59A mutant of MicA, a YycF response-regulator homologue from Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2004Alan Riboldi-Tunnicliffe RR02 (MicA) is an essential bacterial protein that belongs to the YycF family of response regulators and consists of two domains: an N-terminal receiver domain and a C-terminal effector domain. Streptococcus pneumoniae RR02 (MicA; residues 2,234) has been crystallized using the sitting-drop vapour-diffusion technique. The crystals belong to space group P21, with unit-cell parameters a = 46.46, b = 32.61, c = 63.35,Å, , = 90.01°. X-ray diffraction data have been collected to 1.93,Å resolution. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Mitsuru Momma A recombinant form of Arthrobacter globiformis inulin fructotransferase (DFAIII-producing) has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the hanging-drop vapour-diffusion technique using 0.1,M Na HEPES pH 7.5 buffer containing 1.5,M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffracted to better than 1.5,Å at 100,K using a synchrotron-radiation source at the Photon Factory. The crystal belonged to space group R32, with unit-cell parameters a = b = 92.02, c = 229.82,Å in the hexagonal axes. Assuming the presence of one molecule in the asymmetric unit, the VM value for the crystal was 2.15,Å3,Da,1, indicating a solvent content of 42.8%. Selenomethionine-derivative crystals belonged to a different space group, C2, with unit-cell parameters a = 159.32, b = 91.92, c = 92.58,Å, , = 125.06. Matthews coefficient calculations suggested that the C2 selenomethionine-derivative crystal contained three molecules per asymmetric unit. [source] Crystallization and preliminary X-ray data investigation of the bacterial enterocin A immunity protein at 1.65,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2003Bjørn Dalhus Crystals of the bacterial enterocin A immunity protein have been prepared by the hanging-drop vapour-diffusion technique at 293,K. The crystals diffract to better than 1.7,Å resolution and X-ray diffraction data to 1.65,Å have been collected at 110,K using synchrotron radiation. The enterocin A immunity protein crystals belong to the monoclinic crystal system, with unit-cell parameters a = 116.32, b = 42.35, c = 66.17,Å, , = 111.3°. The symmetry and systematic absences in the diffraction pattern are consistent with space group C2. The presence of two molecules in the asymmetric unit with a molecular weight of ,12.2,kDa gives a crystal volume per protein mass (VM) of ,3.1,Å3,Da,1 and a solvent content of ,60% by volume. [source] Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathioneACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2003Katia D'Ambrosio A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55,Å resolution. The crystals belong to space group P212121, with unit-cell parameters a = 45.7, b = 49.1, c = 104.8,Å. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73,Å using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress. [source] Cloning, overexpression, purification, crystallization and preliminary diffraction analysis of the receiver domain of MicAACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2003Colin J. Bent MicA is a response regulator from Streptococcus pneumoniae thought to be involved in redox-energy sensing under oxygen-limiting environments. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. X-ray diffraction data were collected using synchrotron radiation to a resolution of 1.91,Å. The crystals belong to the monoclinic space group C2221, with unit-cell parameters a = 78.69, b = 92.57, c = 37.16,Å, , = , = , = 90.0°. The Matthews coefficient indicates that MicA crystallizes with one molecule in the asymmetric unit. [source] Crystallization and preliminary X-ray analysis of candoxin, a novel reversible neurotoxin from the Malayan krait Bungarus candidusACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Palasingam Paaventhan Candoxin, a novel three-finger toxin from Bungarus candidus, is a reversible antagonist of muscle (,,,,) but a poorly reversible antagonist of neuronal ,7 nicotinic acetylcholine receptors. It has a molecular weight of 7344,Da, with 66 amino-acid residues including ten half-cystines. The fifth disulfide bridge is located at the tip of loop I (Cys6,Cys11) instead of in loop II as found in other ,-neurotoxins. Interestingly, candoxin lacks the segment cyclized by the fifth disulfide bridge at the tip of the middle loop of long-chain neurotoxins, which was reported to be critical for binding to ,7 receptors. As a first step to determining its three-dimensional structure, candoxin was crystallized by the hanging-drop vapour-diffusion technique in conditions around 1.5,M sodium chloride, 10%(v/v) ethanol. The crystals formed belonged to the hexagonal system, space group P6222, with unit-cell parameters a = 54.88, b = 54.88, c = 75.54,Å, , = , = 90, , = 120°, and diffract to a resolution of 1.80,Å. The crystallographic asymmetric unit contains one molecule of candoxin, with an estimated solvent content of 44.6%. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated. [source] Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidusACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-2 2002L. Watanabe Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000,8000,Da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1,M sodium citrate pH 5.6, 15% PEG 4000 and 0.15,M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09,Å, , = 111.32°, and diffract to a nominal resolution of 1.61,Å. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60,Å, , = 125.55°, and diffract to a nominal resolution of 2.78,Å. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated. [source] Purification and crystallization of the yeast elongation factor eEF2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002René Jørgensen Crystals of the Saccharomyces cerevisiae elongation factor 2 (eEF2) in complex with GDP were obtained with the vapour-diffusion technique after rapid purification from industrial yeast. The crystals diffract to 2.85,Å and belong to the space group P212121. A yeast strain expressing a functional histidine-tagged eEF2 as the only form of the protein further allows facilitated purification of the factor for both structural and functional studies. [source] Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeriACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Xavier Carpena Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P21, with unit-cell parameters a = 60.6, b = 153.9, c = 109.2,Å, , = 102.8°. From these crystals a diffraction data set to 1.8,Å resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I212121), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5,Å. These crystals diffracted beyond 2.2,Å resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis. [source] Crystallization and preliminary X-ray diffraction analysis of a [2Fe,2S] ferredoxin (FdVI) from Rhodobacter capsulatusACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2001Jean Armengaud A [2Fe,2S] ferredoxin found in the photosynthetic bacterium Rhodobacter capsulatus has been purified in recombinant form from Escherichia coli. This protein, called FdVI, resembles ferredoxins involved in iron,sulfur cluster biosynthesis in various prokaryotic and eukaryotic cells. Purified recombinant FdVI was recovered in high yields and appeared to be indistinguishable from the genuine R. capsulatus ferredoxin based on UV,visible absorption and EPR spectroscopy and mass spectrometry. FdVI has been crystallized in the oxidized state by a sitting-drop vapour-diffusion technique using sodium formate as precipitant. Seeding larger drops from a previous hanging-drop-grown small crystal resulted in the formation of long red,brown prismatic needles. Preliminary X-ray diffraction analysis indicated that FdVI crystals are orthorhombic and belong to the space group P212121, with unit-cell parameters a = 45.87, b = 49.83, c = 54.29 Å. [source] Crystallization and preliminary crystallographic analysis of N -acetylglucosamine 6-phosphate deacetylase from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2000F. M. Ferreira N -Acetylglucosamine 6-phosphate deacetylase (E.C. 3.5.1.25), an enzyme from Escherichia coli involved in aminosugar catabolism, has been crystallized by the vapour-diffusion technique using phosphate as precipitant. X-ray diffraction experiments show the crystals to belong to the orthorhombic crystal system, with space group P21212. The unit-cell parameters are a = 82.09,(2), b = 114.50,(1), c = 80.17,(1),Å. The crystals diffract to a maximum resolution of 1.8,Å and an initial data set was collected to 2.0,Å. [source] Expression, purification, crystallization and preliminary X-ray crystallographic data from TktA, a transketolase from the lactic acid bacterium Lactobacillus salivariusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Matt Horsham The enzyme transketolase from the lactic acid bacterium Lactobacillus salivarius (subsp. salivarius UCC118) has been recombinantly expressed and purified using an Escherichia coli expression system. Purified transketolase from L. salivarius has been crystallized using the vapour-diffusion technique. The crystals belonged to the trigonal space group P3221, with unit-cell parameters a = b = 75.43, c = 184.11,Å, and showed diffraction to 2.3,Å resolution. [source] Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Rafael Miguez Couñago 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 P6122 or its enantiomorph P6522, with unit-cell parameters a = b = 75.62, c = 282.49,Å, , = 90, , = 90, , = 120°. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-terminal carbohydrate-recognition domain of human galectin-4ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Ana Lucia L. R. Zimbardi Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2,Å resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6122, with unit-cell parameters a = b = 71.25, c = 108.66,Å. The asymmetric unit contained one molecule of hGal4-CRD-1, with a VM value of 2.34,Å3,Da,1 and a solvent content of 47.51%. [source] Expression, crystallization and preliminary crystallographic analysis of RNA-binding protein Hfq (YmaH) from Bacillus subtilis in complex with an RNA aptamerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Seiki Baba 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. [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 X-ray study of the d -altritol oligonucleotide GTGTACACACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Margriet Ovaere In altritol nucleic acids (ANAs), the natural five-membered ribose ring of RNA is replaced by the six-membered d -altritol ring. ANAs are good candidates to act as siRNAs in the RNA-interference pathway. Crystals of the fully modified altritol self-complementary octamer GTGTACAC were grown by the hanging-drop vapour-diffusion technique at 289,K. Diffraction data were recorded on SLS beamline X06DA and processed to 3.0,Å resolution. The crystals belonged to the hexagonal space group P6122 or P6522, with unit-cell parameters a = 25.05, c = 117.58,Å. [source] Preliminary crystallographic characterization of the Grb2 SH2 domain in complex with a FAK-derived phosphotyrosyl peptideACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Hsiao-Hsin Chen Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2,FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921,930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3121, with unit-cell parameters a = b = 102.7, c = 127.6,Å, , = , = 90.0, , = 120.0°. A diffraction data set was collected from a flash-cooled crystal at 100,K to 2.49,Å resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress. [source] Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutansACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Qiao-Ming Hou The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and ,-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6,Å resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1,Å. [source] Purification, crystallization and preliminary X-ray analysis of bifunctional isocitrate dehydrogenase kinase/phosphatase in complex with its substrate, isocitrate dehydrogenase, from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009Jimin Zheng Escherichia coli isocitrate dehydrogenase (ICDH) can be phosphorylated and dephosphorylated by a single bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (AceK), which is encoded by the aceK gene. In order to investigate the regulatory mechanism of (de)phosphorylation of ICDH by AceK, AceK was successfully cocrystallized in complex with its intact protein substrate, ICDH, in the presence of ATP. The complex crystal was obtained by the hanging-drop vapour-diffusion technique using PEG 300 as a precipitant and magnesium sulfate as an additive. SDS,PAGE analysis of dissolved crystals showed that the crystals contained both AceK and ICDH proteins. The complex crystals diffracted to a resolution of 2.9,Å in space group P63, with unit-cell parameters a = b = 196.80, c = 156.46,Å. [source] Expression, purification, crystallization and preliminary X-ray analysis of para -nitrophenol 4-monooxygenase from Pseudomonas putida DLL-E4ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Weidong Liu Para -nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para -nitrophenol by oxidative release of the nitro group from the aromatic ring to form p -benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P212121, with unit-cell parameters a = 54.47, b = 77.56, c = 209.17,Å, and diffracted to 2.24,Å resolution. [source] Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist Trypanosoma cruziACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Kazuaki Matoba Aspartate transcarbamoylase (ATCase), the second enzyme of the de novo pyrimidine-biosynthetic pathway, catalyzes the production of carbamoyl aspartate from carbamoyl phosphate and l -aspartate. In contrast to Escherichia coli ATCase and eukaryotic CAD multifunctional fusion enzymes, Trypanosoma cruzi ATCase lacks regulatory subunits and is not part of the multifunctional fusion enzyme. Recombinant T. cruzi ATCase expressed in E. coli was purified and crystallized in a ligand-free form and in a complex with carbamoyl phosphate at 277,K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. Ligand-free crystals (space group P1, unit-cell parameters a = 78.42, b = 79.28, c = 92.02,Å, , = 69.56, , = 82.90, , = 63.25°) diffracted X-rays to 2.8,Å resolution, while those cocrystallized with carbamoyl phosphate (space group P21, unit-cell parameters a = 88.41, b = 158.38, c = 89.00,Å, , = 119.66°) diffracted to 1.6,Å resolution. The presence of two homotrimers in the asymmetric unit (38,kDa × 6) gives VM values of 2.3 and 2.5,Å3,Da,1 for the P1 and P21 crystal forms, respectively. [source] Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a, a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venomACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Rodrigo Novaes Ferreira Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60,Å resolution were collected from two crystals (free enzyme and the endogenous ligand,protein complex, respectively). They both belonged to space group P212121, with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3,Å for the free-enzyme crystal). [source] Purification, crystallization and preliminary X-ray analysis of FliT, a bacterial flagellar substrate-specific export chaperoneACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Miki Kinoshita The assembly process of the bacterial flagellum is coupled to flagellar gene expression. FliT acts not only as a flagellar type III substrate-specific export chaperone for the filament-capping protein FliD but also as a negative regulator that suppresses flagellar gene expression through its specific interaction with the master regulator FlhD4C2 complex. In this study, FliT of Salmonella enterica serovar Typhimurium was expressed, purified and crystallized. Crystals of SeMet FliT were obtained by the sitting-drop vapour-diffusion technique with potassium/sodium tartrate as the precipitant. The crystals grew in the trigonal space group P3121 or P3221 and diffracted to 3.2,Å resolution. The anomalous difference Patterson map of the SeMet FliT crystal showed significant peaks in its Harker sections, indicating the usefulness of the derivative data for structure determination. [source] Expression, crystallization and preliminary crystallographic analysis of the PAS domain of RsbP, a stress-response phosphatase from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Masatomo Makino RsbP, a regulator of RNA polymerase ,B activity in Bacillus subtilis, is a phosphatase containing a Per,Arnt,Sim (PAS) domain in its N-terminal region that is expected to sense energy stresses such as carbon, phosphate or oxygen starvation. Energy-stress signals are transmitted to the PAS domain and activate the C-terminal phosphatase domain of RsbP, leading to activation of the downstream anti-anti-,B factor RsbV. Finally, the general stress response is induced to protect the cells against further stresses. The recombinant PAS domain of RsbP was crystallized by the sitting-drop vapour-diffusion technique using 40% PEG 400 as a precipitant. The crystals belonged to space group P21, with unit-cell parameters a = 55.2, b = 71.7, c = 60.2,Å, , = 92.1°. Diffraction data were collected to a resolution of 1.6,Å. [source] Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Didem Sutay Kocabas Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8,Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit. [source] Crystallization and preliminary crystallographic characterization of the extrinsic PsbP protein of photosystem II from Spinacia oleraceaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009J. Kohoutová 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 P212121, with unit-cell parameters a = 38.68, b = 46.73, c = 88.9,Å. [source] | ||||||||||