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Crystallization Screening (crystallization + screening)
Selected AbstractsSurface-entropy reduction approaches to manipulate crystal forms of ,-ketoacyl acyl carrier protein synthase II from Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2008Gopalakrishnan Parthasarathy A series of experiments with ,-ketoacyl acyl carrier protein synthase II (FabF) from Streptococcus pneumonia (spFabF) were undertaken to evaluate the capability of surface-entropy reduction (SER) to manipulate protein crystallization. Previous work has shown that this protein crystallizes in two forms. The triclinic form contains four molecules in the asymmetric unit (a.u.) and diffracts to 2.1,Å resolution, while the more desirable primitive orthorhombic form contains one molecule in the a.u. and diffracts to 1.3,Å. The aim was to evaluate the effect of SER mutations that were specifically engineered to avoid perturbing the crystal-packing interfaces employed by the favorable primitive orthorhombic crystal form while potentially disrupting a surface of the protein employed by the less desirable triclinic crystal form. Two mutant proteins were engineered, each of which harbored five SER mutations. Extensive crystallization screening produced crystals of the two mutants, but only under conditions that differed from those used for the native protein. One of the mutant proteins yielded crystals that were of a new form (centered orthorhombic), despite the fact that the interfaces employed by the primitive orthorhombic form of the native protein were specifically unaltered. Structure determination at 1.75,Å resolution reveals that one of the mutations, E383A, appears to play a key role in disfavouring the less desirable triclinic crystal form and in generating a new surface for a packing interaction that stabilizes the new crystal form. [source] A procedure for setting up high-throughput nanolitre crystallization experiments.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2005Crystallization workflow for initial screening, automated storage, imaging, optimization Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100,nl of protein plus 100,nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294,K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277,K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported. [source] Preparation and preliminary X-ray analysis of the catalytic module of ,-1,3-xylanase from the marine bacterium Vibrio sp.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004,-1,3-xylanase (1,3-,- d -xylan xylanohydrolase; EC 3.2.1.32) is an enzyme capable of hydrolyzing ,-1,3-xylan. The newly cloned ,-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 (XYL4) exhibited a modular structure consisting of three modules: an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two C-terminal xylan-binding modules belonging to carbohydrate-binding module family 31. Despite substantial crystallization screening, crystallization of the recombinant XYL4 was not accomplished. However, the deletion mutant of XYL4, composed of a catalytic module without a xylan-binding module, was crystallized. The crystal belonged to space group P212121, with unit-cell parameters a = 51.6, b = 75.8, c = 82.0,Å. X-ray diffraction data were collected to 1.44,Å resolution. [source] When less is more: a more efficient vapour-diffusion protocolACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003Kirsty V. Dunlop Reducing protein consumption during crystallization screening is of utmost importance to crystallographers because of the time, effort and money that goes into producing pure protein. One approach is to reduce sample volumes with robotics, but a patent and the high cost of equipment limits access. Here, it is shown that the same result can be obtained by reducing the sample concentration in a modified vapour-diffusion protocol, the dilution method. In this protocol, the protein and mother liquor in the crystallization drop are both diluted, while the mother liquor in the well remains undiluted. Vapour diffusion will shrink the initial volume of the crystallization drop, e.g. 1,µl or more, to a drop size equivalent to one dispensed by a robot. This new crystallization method circumvents some of the current problems associated with robotic crystallization screening trials. Because of the large initial volume of the crystallization drop, the evaporation problem is eliminated and dispensing accuracy is improved. In addition, the likelihood that the crystallization experiment starts in the undersaturated region is increased. [source] Purification and crystallization of the human RXR, ligand-binding domain,9- cisRA complexACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Pascal F. Egea The purification and crystallization of the stoichiometric complex of human RXR, ligand-binding domain (hRXR, LBD) bound to its natural ligand 9- cis retinoic acid (9- cisRA) are described. A three-step purification yields a pure and homogenous complex. Based on the crystallization conditions of several other nuclear receptors, an exhaustive crystallization screening using carboxylic acids as precipitating agents was performed in association with the use of polyhydric alcohols acting as cosmotropic solutes. Crystals of the hRXR, LBD,9- cisRA complex grew in a tripartite mixture containing sodium formate, glycerol and propane-1,2-diol. Micro- and macroseeding were necessary to improve both the size and the quality of crystals in order to make them suitable for structure determination. [source] Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 proteinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Tom J. Petty Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ,3.2,Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74,Å. Structure determination is ongoing. [source] Laser-improved protein crystallization screeningACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Neela Yennawar Screening of proteins for crystallization under laser irradiation was investigated using six proteins: ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose dehydrogenase and myoglobin. Shining 532,nm green circularly polarized laser light with a picosecond pulse and 6,mW power for 30,s on newly set-up protein drops showed a marked improvement in the number of screen conditions amenable for crystal growth compared with control drops under identical conditions but without laser exposure. For glucose dehydrogenase and sorbitol dehydrogenase, larger and better quality crystals were formed and the resolution of X-ray diffraction was improved. The speed of crystallization increased in the case of ribonuclease B, lysozyme and sorbitol dehydrogenase. During laser irradiation, the amount of precipitation in the screened drops increased, indicating a transient decrease in protein solubility. At the optimized laser settings, there was no deleterious effect of the laser on crystal growth or on the protein. In the cases of ribonuclease B and lysozyme the crystal packing did not change owing to the laser exposure. [source] Crystallization and preliminary X-ray analysis of a phosphopentomutase from Bacillus cereusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Timothy D. Panosian Phosphopentomutases (PPMs) interconvert d -ribose 5-phosphate and ,- d -ribose 1-phosphate to link glucose and nucleotide metabolism. PPM from Bacillus cereus was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Bacterial PPMs are predicted to contain a di-metal reaction center, but the catalytically relevant metal has not previously been identified. Sparse-matrix crystallization screening was performed in the presence or absence of 50,mM MnCl2. This strategy resulted in the formation of two crystal forms from two chemically distinct conditions. The crystals that formed with 50,mM MnCl2 were more easily manipulated and diffracted to higher resolution. These results suggest that even if the catalytically relevant metal is not known, the crystallization of putative metalloproteins may still benefit from supplementation of the crystallization screens with potential catalytic metals. [source] Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chainsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Daichi Morimoto Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8,Å resolution, respectively. The tetraubiquitin crystals belonged to space group C2221, with unit-cell parameters a = 58.795, b = 76.966, c = 135.145,Å. The hexaubiquitin crystals belonged to space group P21, with unit-cell parameters a = 51.248, b = 102.668, c = 51.161,Å. Structural analysis by molecular replacement is in progress. [source] | ||||||||||