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Sitting-drop Vapor Diffusion (sitting-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] Crystallization and initial X-ray diffraction of BtuB, the integral membrane cobalamin transporter of Escherichia coliACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003David P. Chimento BtuB, the cobalamin transporter from Escherichia coli, has been overexpressed, purified and crystallized. The purified protein was solubilized in n -octyl tetraoxyethylene (C8E4) and was crystallized using sitting-drop vapor diffusion with PEG 3350 and magnesium acetate as precipitants (pH 6.5). Two crystal forms have been obtained. Crystal type I belongs to space group P3121, with unit-cell parameters a = b = 81.6, c = 210.0,Å, , = , = 90, , = 120°. Crystal type II belongs to space group P3121, with unit-cell parameters a = b = 81.6, c = 226.0,Å, , = , = 90, , = 120°. Each crystal form contains a monomer in the asymmetric unit. Diffraction for crystal type I extends to 2.0,Å and diffraction for crystal type II extends to 2.7,Å. Both crystal forms are suitable for structure determination. [source] Purification, crystallization and preliminary X-ray analysis of Caenorhabditis elegans ubiquitin-conjugation enzyme M7.1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003José A. Gavira M7.1 is a class IV ubiquitin-conjugation enzyme (UBC) that belongs to the ubiquitination cascade in Caenorhabditis elegans. The clone for this UBC has been overexpressed in Escherichia coli and the 16.7,kDa protein was purified from the soluble fraction. M7.1 was crystallized by sitting-drop vapor diffusion in 10% ethanol, 1.5,M NaCl at 277.5,K. Crystals diffracted to 1.75,Å and belong to the orthorhombic space group P212121, with unit-cell parameters a = 44.3, b = 54.3, c = 60.2,Å. The asymmetric unit contains a single monomer. A molecular-replacement model has been determined and refinement is in progress. [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] Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNPACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Mark Del Campo The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/,598,664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (,1,mg,ml,1), but its solubility could be increased by adding 50,mMl -arginine plus 50,mMl -glutamate and 50% glycerol to achieve concentrations of ,10,mg,ml,1. Initial crystals were obtained by the microbatch method in the presence of a U10 RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/,598,664 in complex with AMP-PNP and U10 belonged to space group P21212, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52,Å, and diffracted X-rays to beyond 1.9,Å resolution using synchrotron radiation from sector 21 at the Advanced Photon Source. [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] | ||||||||||