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Crystallization Screens (crystallization + screen)
Selected AbstractsCrystallizing proteins on the basis of their precipitation diagram determined using a microfluidic formulatorJOURNAL OF SYNCHROTRON RADIATION, Issue 6 2005Morten O. A. Sommer Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10,µL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials. [source] A crystallization screen based on alternative polymeric precipitantsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Clemens Grimm Most commercially available crystallization screens are sparse-matrix screens with a predominance of inorganic salts and polyethylene glycols (PEGs) as precipitants. It was noted that commercially available screens are largely unsatisfactory for the purpose of the crystallization of multimeric protein and protein,nucleic acid complexes. This was reasoned to be a consequence of the redundancy in screening crystallization parameter space by the predominance of PEG as a precipitant in standard screens and it was suggested that this limitation could be overcome by introducing a variety of other organic polymers. Here, a set of 288 crystallization conditions was devised based on alternative polymeric precipitants and tested against a set of 20 different proteins/complexes; finally, a screen comprising the 96 most promising conditions designed to complement PEG- and salt-based commercial screens was proposed. [source] Crystallizing proteins on the basis of their precipitation diagram determined using a microfluidic formulatorJOURNAL OF SYNCHROTRON RADIATION, Issue 6 2005Morten O. A. Sommer Crystallization of proteins from a purified protein solution remains a bottleneck in the structure determination pipeline. In this paper the crystallization problem is addressed using a microfluidic device capable of determining detailed protein precipitation diagrams using less than 10,µL of protein sample. Based on the experimentally determined protein phase behavior, a crystallization screen can be designed to accommodate the physical chemistry of the particular protein target. Such a tailor-made crystallization screen has a high probability of yielding crystallization hits. The approach is applied to two different proteins: the calcium pump (SERCA), an eukaryotic integral membrane protein, and UMP kinase, a prokaryotic soluble kinase. Protein phase behavior is mapped for both proteins and tailor-made crystallization screens are designed for the two proteins resulting in about 50% crystallization probability per experiment. This illustrates the power of using microfluidic devices for detailed characterization of protein phase behavior prior to crystallization trials. [source] A crystallization screen based on alternative polymeric precipitantsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010Clemens Grimm Most commercially available crystallization screens are sparse-matrix screens with a predominance of inorganic salts and polyethylene glycols (PEGs) as precipitants. It was noted that commercially available screens are largely unsatisfactory for the purpose of the crystallization of multimeric protein and protein,nucleic acid complexes. This was reasoned to be a consequence of the redundancy in screening crystallization parameter space by the predominance of PEG as a precipitant in standard screens and it was suggested that this limitation could be overcome by introducing a variety of other organic polymers. Here, a set of 288 crystallization conditions was devised based on alternative polymeric precipitants and tested against a set of 20 different proteins/complexes; finally, a screen comprising the 96 most promising conditions designed to complement PEG- and salt-based commercial screens was proposed. [source] Effects of impurities on membrane-protein crystallization in different systemsACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Christopher A. Kors When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein. [source] Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehydeACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Matthew W. Vetting The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane,dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6,Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68,Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups. [source] First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical valueACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006L. Banci The EC `Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, ,170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples. [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] | ||||||||||