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Diffracting Crystals (diffracting + crystal)
Selected AbstractsCrystallization and preliminary crystallographic analysis of the ADP-ribosyltransferase HopU1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Yan Lin Several Gram-negative pathogens of plants and animals and some eukaryotic associated bacteria use type III protein-secretion systems (T3SSs) to deliver bacterial virulence-associated `effector' proteins directly into host cells. HopU1 is a type III effector protein from the plant pathogen Pseudomonas syringae, which causes plant bacterial speck disease. HopU1 quells host immunity through ADP-ribosylation of GRP7 as a substrate. HopU1 has been reported as the first ADP-ribosyltransferase virulence protein to be identified in a plant pathogen. Although several structures of ADP-ribosyltransferases have been determined to date, no structure of an ADP-ribosyltransferase from a plant pathogen has been determined. Here, the protein expression, purification, crystallization and preliminary crystallographic analysis of HopU1 are reported. Diffracting crystals were grown by hanging-drop vapour diffusion using polyethylene glycol 10,000 as a precipitant. Native and SAD data sets were collected using native and selenomethionine-derivative HopU1 crystals. The diffraction pattern of the crystal extended to 2.7,Å resolution using synchrotron radiation. The crystals belonged to space group P43, with unit-cell parameters a = 92.6, b = 92.6, c = 101.6,Å. [source] Crystallization and preliminary crystallographic analysis of nosiheptide-resistance methyltransferase from Streptomyces actuosus in complex with SAMACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Huirong Yang Nosiheptide-resistance methyltransferase (NSR) methylates 23S rRNA at the nucleotide adenosine 1067 in Escherichia coli and thus contributes to resistance against nosiheptide, a sulfur-containing peptide antibiotic. Here, the expression, purification and crystallization of NSR from Streptomyces actuosus are reported. Diffracting crystals were grown by the hanging-drop vapour-diffusion method in reservoir solution consisting of 0.35,M ammonium chloride, 24%(w/v) PEG 3350, 0.1,M MES pH 5.7 at 293,K. Native data have been collected from the apo enzyme and a SAM complex, as well as apo SeMet SAD data. The diffraction patterns of the apo form of NSR, of NSR complexed with SAM and of SeMet-labelled NSR crystals extended to 1.90, 1.95 and 2.25,Å resolution, respectively, using synchrotron radiation. All crystals belonged to space group P21, with approximate unit-cell parameters a = 64.6, b = 69.6, c = 64.9,Å, , = 117.8°. [source] Crystallization of dihydrodipicolinate synthase from a clinical isolate of Streptococcus pneumoniaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Natalia E. Sibarani Dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) catalyzes the rate-limiting step in the (S)-lysine biosynthesis pathway of bacteria and plants. Here, the cloning of the DHDPS gene from a clinical isolate of Streptococcus pneumoniae (OXC141 strain) and the strategy used to express, purify and crystallize the recombinant enzyme are described. Diffracting crystals were grown in high-molecular-weight PEG precipitants using the hanging-drop vapour-diffusion method. The best crystal, from which data were collected, diffracted to beyond 2.0,Å resolution. Initially, the crystals were thought to belong to space group P42212, with unit-cell parameters a = 105.5, b = 105.5, c = 62.4,Å. However, the R factors remained high following initial processing of the data. It was subsequently shown that the data set was twinned and it was thus reprocessed in space group P2, resulting in a significant reduction in the R factors. Determination of the structure will provide insight into the design of novel antimicrobial agents targeting this important enzyme from S. pneumoniae. [source] Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigasACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Shabir Najmudin The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12,kDa protein that contains a novel mixed-metal sulfide cluster of the type [S2MoS2CuS2MoS2]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25,Å resolution in-house and to beyond 2.0,Å resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106,Å. [source] Elastic deformations in a perfect bulk Si crystal studied by high-energy X-raysJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 5 2009Alexander Gröschel Long-range strain fields induced in highly perfect bulk crystals during the manufacturing process significantly affect the quality and may even lead to spontaneous fracturing. Obviously a quantitative assessment of these deformations is crucial. A possible means is to examine the diffraction of X-rays by strained crystals, as the deformations bear on the diffraction characteristics of such crystals. In this report a quantitative examination of the diffraction characteristics of a perfect silicon bulk crystal with long-range strain fields in a well defined geometry is presented. The experiments were carried out using a high-energy X-ray laboratory source. By simulating the elastic deformation of the crystal by a finite element program the strain fields of the diffracting crystal are accessed. From these, simulated data values for integrated intensities can be derived on the basis of the dynamical diffraction theory for slightly distorted crystals. The theoretical calculations show good agreement with the experimental measured values. The smallest deformation yielding a noticeable change of the integrated intensity can be associated with a bending radius of the diffracting lattice planes of 16,km. [source] Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Lu Lu Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the ,-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289,K. The crystal diffracted to 1.6,Å resolution using synchrotron radiation and belonged to space group I41, with unit-cell parameters a = 55.0, b = 55.0, c = 67.4,Å. [source] Comparative refinement of correct and incorrect structural models of tetrabutylammonium tetrabutylborate , pitfalls arising from poor-quality dataACTA CRYSTALLOGRAPHICA SECTION A, Issue 4 2010Vladimir Stilinovi This paper demonstrates how numerical parameters usually used to assess the quality of a crystal structure solution (R, wR and S) may be misleading when studying a model refined against poor-quality data. Weakly diffracting crystals of tetrabutylammonium tetrabutylborate, a low-density organic salt comprising isoelectronic cations and anions, were measured using Cu and Mo K, radiation. Along with the correct structural model, six erroneous structural models were constructed and refined against the same data. For both data sets it was found that models based on an incorrect unit-cell choice give lower values of R and wR than the correct one, thus apparently being in better agreement with measured data. Closer inspection of the measured data shows that this is in fact not the case. [source] A rational approach to heavy-atom derivative screeningACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010M. Gordon Joyce Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the `classical' heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom compound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the process of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing. [source] The interdependence of wavelength, redundancy and dose in sulfur SAD experimentsACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2008Michele Cianci In the last decade, the popularity of sulfur SAD anomalous dispersion experiments has spread rapidly among synchrotron users as a quick and streamlined way of solving the phase problem in macromolecular crystallography. On beamline 10 at SRS (Daresbury Laboratory, UK), a versatile design has allowed test data sets to be collected at six wavelengths between 0.979 and 2.290,Å in order to evaluate the importance and the interdependence of experimental variables such as the Bijvoet ratio, wavelength, resolution limit, data redundancy and absorbed X-ray dose in the sample per data set. All the samples used in the experiments were high-quality hen egg-white lysozyme crystals. X-radiation damage was found to affect disulfide bridges after the crystals had been given a total dose of 0.20 × 107,Gy. However, with such a total dose, it was still possible in all cases to find a strategy to collect data sets to determine the sulfur substructure and produce good-quality phases by choosing an optimum combination of wavelength, exposure time and redundancy. A ,|,ano|/,(,ano), greater than 1.5 for all resolution shells was a necessary requirement for successful sulfur SAD substructure location. Provided this is achieved, it seems possible to find an optimum compromise between wavelength, redundancy and dose to provide phasing information. The choice of the wavelength should then follow the sample composition and the diffracting properties of the crystal. For strongly diffracting crystals, wavelengths equal or shorter than 1.540,Å can be selected to capture the available data (provided the Bijvoet ratio is reasonable), while a longer wavelength, to gain as high a Bijvoet ratio as possible, must be used for more weakly diffracting crystals. These results suggest that an approach to a sulfur SAD experiment based on a complete description of the crystal system and the instrument for data collection is useful. [source] High-resolution diffracting crystals of intrinsically active p38, MAP kinase: a case study for low-throughput approachesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2007Ron Diskin p38 MAP kinases are central signalling molecules that mediate cellular responses to numerous environmental conditions and signalling molecules. Their proper function is required for many processes, including stress response, apoptosis, differentiation, growth and even learning and memory. Abnormal activity of p38 MAP kinases is associated with the aetiology of many diseases, making understanding their activation mechanisms highly critical. In this respect, mechanistic insights may be derived from structural studies of recently developed intrinsically active p38, mutants. Unlike wild-type p38,, which routinely crystallized, the active mutants caused severe difficulties during the crystallization process. The main hindrance was found to be protein heterogeneity, which was meticulously resolved by genetically modifying the recombinant protein and optimizing the expression and purification protocols. The success in obtaining crystallizable proteins strongly emphasizes that in certain cases, high-throughput techniques (crystallization robots) together with low-throughput approaches, with careful monitoring and analysis of the results, are essential. [source] Crystallization and preliminary X-ray crystallographic studies on the fungal immunomodulatory protein Fve from the golden needle mushroom (Flammulina velutipes)ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2003See Voon Seow The fungal immunomodulatory protein from the edible golden needle mushroom (Flammulina velutipes), designated Fve, is a single polypeptide consisting of 114 amino-acid residues. It is believed to trigger the mitogenic proliferation of T lymphocytes and Th1 cytokine production. Here, it is demonstrated that Fve forms a homodimer in nature. In order to understand the relationship between its structure and function, Fve was crystallized using the hanging-drop method; the protein formed well diffracting crystals within 3,5,d in 2.5% PEG 400, 2.0,M ammonium sulfate and 0.1,M Tris base buffer pH 8.5. The space group of the Fve crystals is either P43212 or P41212, with unit-cell parameters a = b = 96.92, c = 61.42,Å. The crystal contains two molecules per asymmetric unit and diffracts to 1.4,Å resolution when exposed to synchrotron radiation. [source] Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding proteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2002Matthew H. Bucher Pyrococcus furiosus maltodextrin-binding protein readily forms large orthorhombic crystals that diffract to high resolution. This protein was used as a model system to investigate the influence of five short affinity tags (His6, Arg5, Strep tag II, FLAG tag and the biotin acceptor peptide) on the formation of protein crystals and their ability to diffract X-rays. The results indicate that the amino-acid sequence of the tag can have a profound effect on both of these parameters. Consequently, the ability to obtain diffracting crystals of a particular protein may depend as much on which affinity tag is selected as it does on whether an affinity tag is used at all. [source] Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010César Santiago The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H,CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method. [source] Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009Stefan Kernstock Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K+ and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9,Å. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K+ in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties. [source] Improvement of the quality of lumazine synthase crystals by protein engineeringACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2008Lidia Rodríguez-Fernández Icosahedral macromolecules have a wide spectrum of potential nanotechnological applications, the success of which relies on the level of accuracy at which the molecular structure is known. Lumazine synthase from Bacillus subtilis forms a 150,Å icosahedral capsid consisting of 60 subunits and crystallizes in space group P6322 or C2. However, the quality of these crystals is poor and structural information is only available at 2.4,Å resolution. As classical strategies for growing better diffracting crystals have so far failed, protein engineering has been employed in order to improve the overexpression and purification of the molecule as well as to obtain new crystal forms. Two cysteines were replaced to bypass misfolding problems and a charged surface residue was replaced to force different molecular packings. The mutant protein crystallizes in space group R3, with unit-cell parameters a = b = 313.02, c = 365.77,Å, , = , = 90.0, , = 120°, and diffracts to 1.6,Å resolution. [source] Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteinsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006Tarmo P. Roosild The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the `soluble' portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K+ channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86,Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments. [source] | ||||||||||