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Sitting-drop Vapour-diffusion Technique (sitting-drop + 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] 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] 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 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 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] 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] 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] 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] 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 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] Expression, purification, crystallization and preliminary X-ray diffraction analysis of the transcriptional repressor SirR from Mycobacterium tuberculosis H37RvACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Baisakhee Saha SirR, a metal-dependent transcriptional repressor from Mycobacterium tuberculosis (Rv2788), was cloned in pQE30 expression vector with an N-terminal His6 tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P41212/P43212, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85,Å. The X-ray diffraction data were processed to a maximum resolution of 2.5,Å. The Matthews coefficient suggests the presence of two (VM = 4.01,Å3,Da,1) to four (VM = 2.0,Å3,Da,1) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (, = 90°) and also a twofold symmetry axis around the z axis (, = 180°). [source] Crystallization and preliminary X-ray analysis of a C-terminal fragment of FlgJ, a putative flagellar rod cap protein from SalmonellaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Yuki Kikuchi The formation of the bacterial flagellar axial structure, including the filament, the hook and the rod, requires the attachment of a cap complex to the distal end of the growing structure. Because the rod penetrates the peptidoglycan (PG) layer, the rod cap complex is thought to have PG-hydrolyzing activity. FlgJ is a putative rod cap protein whose C-terminal region shows sequence similarity to known muramidases. In this study, FlgJ120,316, a C-terminal fragment of FlgJ which contains the muramidase region, was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 3350 as a crystallizing agent and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 38.8, b = 43.9, c = 108.5,Å. Anomalous difference Patterson maps calculated from the diffraction data set of a selenomethionine-labelled crystal showed significant peaks in the Harker sections, indicating that the data were suitable for structure determination. [source] Crystallization and preliminary X-ray analysis of FliJ, a cytoplasmic component of the flagellar type III protein-export apparatus from Salmonella sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Tatsuya Ibuki The axial component proteins of the bacterial flagellum are synthesized in the cytoplasm and then translocated into the central channel of the flagellum by the flagellar type III protein-export apparatus for self-assembly at the distal growing end of the flagellum. FliJ is an essential cytoplasmic component of the export apparatus. In this study, Salmonella FliJ with an extra three residues (glycine, serine and histidine) attached to the N-terminus as the remainder of a His tag (GSH-FliJ) was purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 300 as a precipitant. GSH-FliJ crystals grew in the hexagonal space group P6122 or P6522. While the native crystals diffracted to 3.3,Å resolution, the diffraction resolution limit of mercury derivatives was extended to 2.1,Å. Anomalous and isomorphous difference Patterson maps of the mercury-derivative crystal showed significant peaks in their Harker sections, indicating the usefulness of the derivative data for structure determination. [source] Expression and purification of F-plasmid RepE and preliminary X-ray crystallographic study of its complex with operator DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2007Chieko Wada The replication initiator factor RepE of the F plasmid in Escherichia coli is an essential protein that stringently regulates the F-plasmid copy number. The RepE protein has a dual function: its monomer functions as a replication initiator, while its dimer acts as a transcriptional repressor of the repE gene. The wild-type dimeric RepE protein was expressed as an N-terminal histidine-tagged protein, purified under native conditions with a high salt concentration and crystallized in complex with the repE operator DNA using the sitting-drop vapour-diffusion technique. The crystals diffracted to a resolution of 3.14,Å after the application of dehydration and crystal annealing and belong to space group P21, with unit-cell parameters a = 60.73, b = 99.32, c = 95.00,Å, , = 108.55°. [source] Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2005Daniel Ken Inaoka Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD,orotate complex were grown at 277,K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8,Å resolution using synchrotron radiation (, = 0.900,Å). X-ray diffraction data were collected at 100,K and processed to 1.9,Å resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27,Å. The presence of two molecules in the asymmetric unit (2 × 34,kDa) gives a crystal volume per protein weight (VM) of 2.2,Å3,Da,1 and a solvent content of 44%. [source] | ||||||||||