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Matthews Coefficient VM (Matthew + coefficient_vm)
Selected AbstractsCrystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex formsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Teruya Nakamura During replication, Escherichia coli MutT prevents the misincorporation of mutagenic 8-oxoguanine into nascent DNA strands opposite adenine by hydrolyzing 8-oxo-dGTP in nucleotide pools to 8-oxo-dGMP. E. coli MutT is the most widely investigated member of the Nudix hydrolase family, which is large and found in all organisms. By co-crystallization of MutT with 8-oxo-dGMP, a reaction product, crystals of the binary complex were obtained using ammonium sulfate as a precipitant. The crystals belong to space group P212121, with unit-cell parameters a = 37.9, b = 56.0, c = 59.4,Å. Assuming the presence of one protein,nucleotide complex in the asymmetric unit, the Matthews coefficient VM is 2.1,Å3,Da,1. Crystals of the ternary complex were prepared by soaking crystals of the binary complex in 1,mM MnCl2 solution. They diffracted to 1.96 and 2.56,Å resolution, respectively. [source] Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Paul T. Harris Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25,Å resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79,Å. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8,Å3,Da,1, corresponding to 55.2% solvent content. [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] 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] Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsHACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Kannan Raghunathan EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71,Å resolution. The crystals belonged to space group P212121, with unit-cell parameters a = 53.39, b = 71.11, c = 84.64,Å. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient VM of 2.1,Å3,Da,1, corresponding to 41.5% solvent content. [source] Cloning, purification, crystallization and preliminary crystallographic analysis of a ribokinase from Staphylococcus aureusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Lin Wang The gene SA239 from Staphylococcus aureus encodes a ribokinase that catalyzes the phosphorylation of d -ribose to produce ribose-5-phosphate. Sa239 was crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.9,Å resolution and belonged to space group P6122 or P6522, with unit-cell parameters a = b = 91.8, c = 160.7,Å. Preliminary crystallographic analysis revealed that the Matthews coefficient VM was 3.01,Å3,Da,1, indicating the presence of one molecule in the asymmetric unit. [source] Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of ,-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Kim-Hung Huynh The bacterial ,-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05,Å resolution and belonged to the orthorhombic space group P21212, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3,Å. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.27,Å3,Da,1 and a solvent content of 45.8%. [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] Site-specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Takanori Muto Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS,PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of l -glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35,Å resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 84.0, b = 97.5, c = 108.1,Å, , = 93.0°. Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient VM was calculated to be 3.5,Å3,Da,1 and the solvent content was 65%. [source] Crystallization and preliminary X-ray analysis of the tumour necrosis factor ,,tumour necrosis factor receptor type 2 complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Yohei Mukai Tumour necrosis factor receptor type 2 (TNFR2, TNFRSF1B) is an essential receptor for various host-defence functions of tumour necrosis factor , (TNF). As part of studies to determine the structure of TNFR2, the formation, crystallization and preliminary X-ray diffraction analysis of the TNF,TNFR2 complex are described. The TNF,TNFR2 complex, which comprises one TNF trimer and three TNFR2 monomers, was confirmed and purified by size-exclusion chromatography. Crystals of the TNF,TNFR2 complex were obtained using polyethylene glycol 3350 as a precipitant. The crystal belonged to space group P212121, with unit-cell parameters a = 74.5, b = 117.4, c = 246.8,Å. Assuming the presence of two TNF,TNFR2 complexes in the asymmetric unit, the Matthews coefficient VM was 2.49,Å3,Da,1 and the solvent content of the crystal was 50.7%. The crystal diffracted to 2.95,Å resolution. [source] Preliminary X-ray crystallographic analysis of ornithine acetyltransferase (Rv1653) from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009R. Sankaranarayanan The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N -acetylornithine to l -glutamate to produce N -acetylglutamate and l -ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7,Å and belonged to space group P212121, with unit-cell parameters a = 60.1, b = 99.7, c = 155.3,Å. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient VM of 2.8,Å3,Da,1. [source] Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2007Takanori Muto Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS,PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293,K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30,Å resolution using synchrotron radiation and belong to the trigonal space group P3121, with unit-cell parameters a = b = 92.4, c = 114.3,Å. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient VM was calculated to be 2.5,Å3,Da,1 and the solvent content was 51%. [source] | ||||||||||