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Hanging-drop Vapour-diffusion Technique (hanging-drop + vapour-diffusion_technique)
Selected AbstractsPurification, 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] 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] 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] 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] 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 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] 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] 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] 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] Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2006Francisca Sevilla A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6,kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8,Å from a single crystal flash-cooled at 100,K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23,Å, , = 102.90, , = 104.40, , = 99.07°, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress. [source] Cloning, expression, purification, crystallization and preliminary X-ray studies of epoxide hydrolases A and B from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2006Bichitra K. Biswal Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1,Å resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA. [source] Cloning, expression, purification, crystallization and preliminary crystallographic study of the protein module (BIV2-Helix) in the fusion core of bovine immunodeficiency-like virus (BIV) gp40ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Xiaodong Zhao The fusion core of bovine immunodeficiency virus (BIV) gp40 is proposed to be involved in membrane fusion. However, no crystal structures are yet available. A predicted protein module BIV2-Helix of BIVgp40 has been expressed in Escherichia coli and purified by chromatography. Recombinant BIV2-Helix was crystallized using the hanging-drop vapour-diffusion technique at 291,K. The crystals were grown in MPD and belonged to the primitive rhombohedral space group R3, with unit-cell parameters a = 39.17, b = 39.17, c = 295.05,Å and two molecules per asymmetric unit. X-ray diffraction data were collected to 1.76,Å in the home laboratory from a single crystal. [source] | ||||||||||