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Cryocooled Crystals (cryocooled + crystal)
Selected AbstractsAtomic resolution structure of Escherichia coli dUTPase determined ab initioACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001A. González Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05,Å resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45,Å from a native crystal were also collected and the 100,K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor,substrate-analogue complexes of this protein at very high resolution. [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] Pressure-induced high-density amorphous ice in protein crystalsJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 1 2008Chae Un Kim Crystal cryocooling has been used in X-ray protein crystallography to mitigate radiation damage during diffraction data collection. However, cryocooling typically increases crystal mosaicity and often requires a time-consuming search for cryoprotectants. A recently developed high-pressure cryocooling method reduces crystal damage relative to traditional cryocooling procedures and eases or eliminates the need to screen for cryoprotectants. It has been proposed that the formation of high-density amorphous (HDA) ice within the protein crystal is responsible for the excellent diffraction quality of the high-pressure cryocooled crystals. This paper reports X-ray data that confirm the presence of HDA ice in the high-pressure cryocooled protein crystallization solution and protein crystals analyzed at ambient pressure. Diffuse scattering with a spacing characteristic of HDA ice is seen at low temperatures. This scattering then becomes characteristic successively to low-density amorphous, cubic and hexagonal ice phases as the temperature is gradually raised from 80 to 230,K, and seems to be highly correlated with the diffraction quality of crystals. [source] Crystallization and preliminary X-ray diffraction analyses of the homodimeric glycine decarboxylase (P-protein) from the cyanobacterium Synechocystis sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010PCC 680 Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C1 metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215,kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1,Å were obtained using the hanging-drop vapour-diffusion method at 291,K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P212121, with unit-cell parameters a = 96.30, b = 135.81, c = 179.08,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] | ||||||||||