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Hanging-drop Vapor Diffusion (hanging-drop + vapor_diffusion)
Selected AbstractsA simple technique to convert sitting-drop vapor diffusion into hanging-drop vapor diffusion by solidifying the reservoir solution with agaroseJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2009Tae Woong Whon A simple protocol to convert sitting-drop vapor-diffusion plating into a hanging-drop vapor-diffusion experiment in protein crystallization is reported. After making a sitting-drop plate, agarose solution was added to solidify the reservoir solution, and the plates were incubated upside down. Crystallization experiments with hen egg white lysozyme, thaumatin and glucose isomerase showed that the `upside-down sitting-drop' method could produce single crystals with all the benefits of the hanging-drop crystallization method. [source] Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimuriumACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Mariya V. Dontsova The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9,Å resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P61(5), with unit-cell parameters a = 92.3, c = 267.5,Å. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit. [source] Crystallization and initial crystallographic analysis of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosaACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Catherine A. Regni The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) catalyzes the conversion of mannose 6-phosphate to mannose 1-phosphate in the second step of the alginate biosynthetic pathway of Pseudomonas aeruginosa. PMM/PGM has been crystallized by hanging-drop vapor diffusion in space group P212121. Crystals diffract to 1.75,Å resolution on a synchrotron X-ray source under cryo-cooling conditions. PMM/PGM substituted with selenomethionine has been purified and crystallizes isomorphously with the native enzyme. Structure determination by MAD phasing is under way. Because of its role in alginate biosynthesis, PMM/PGM is a potential target for therapeutic inhibitors to combat P. aeruginosa infections. [source] Crystallization and initial crystallographic analysis of phosphoglucosamine mutase from Bacillus anthracisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Ritcha Mehra-Chaudhary The enzyme phosphoglucosamine mutase catalyzes the conversion of glucosamine 6-phosphate to glucosamine 1-phosphate, an early step in the formation of the nucleotide sugar UDP- N -acetylglucosamine, which is involved in peptidoglycan biosynthesis. These enzymes are part of the large ,- d -phosphohexomutase enzyme superfamily, but no proteins from the phosphoglucosamine mutase subgroup have been structurally characterized to date. Here, the crystallization of phosphoglucosamine mutase from Bacillus anthracis in space group P3221 by hanging-drop vapor diffusion is reported. The crystals diffracted to 2.7,Å resolution under cryocooling conditions. Structure determination by molecular replacement was successful and refinement is under way. The crystal structure of B. anthracis phosphoglucosamine mutase should shed light on the substrate-specificity of these enzymes and will also serve as a template for inhibitor design. [source] Overexpression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae EpsGACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Jason Jens EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6,mM zinc sulfate, 60,mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26,Å and belonged to space group P21, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54,Å. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the regulator AcrR from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2006Ming Li This paper describes the cloning, expression, purification and preliminary X-ray data analysis of the AcrR regulatory protein. The Escherichia coli AcrR is a member of the TetR family of transcriptional regulators. It regulates the expression of the AcrAB multidrug transporter. Recombinant AcrR with a 6×His tag at the C-terminus was expressed in E. coli and purified by metal-affinity chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted to 2.5,Å. The space group was determined to be P32, with unit-cell parameters a = b = 46.61, c = 166.16,Å. [source] | ||||||||||