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Acta Crystallographica Section F: Structural Biology and Crystallization Communications
Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 protein
Wed, 07/28/2010 - 23:00Of 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.
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Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.
Wed, 07/28/2010 - 23:0011S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to ∼80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0–40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 Å resolution and belonged to space group P63, with unit-cell parameters a = b = 97.6, c = 74.8 Å, γ = 120.0°. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress.
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Crystallization and preliminary X-ray crystallographic studies of omega-transaminase from Vibrio fluvialis JS17
Wed, 07/28/2010 - 23:00Omega-transaminase (ω-TA) catalyzes the transfer of an amino group from a non-α-position amino acid or an amine compound with no carboxylic group to an amino acceptor. ω-TA from Vibrio fluvialis JS17 (ω-TAVf) is a novel amine:pyruvate transaminase that is capable of stereoselective transamination of aryl chiral amines. In this study, ω-TAVf was overexpressed in Escherichia coli with engineered C-terminal His tags. ω-TAVf was then purified to homogeneity and crystallized at 292 K. X-ray diffraction data were collected to a resolution of 2.5 Å from a crystal belonging to the orthorhombic space group P212121, with unit-cell parameters a = 78.43, b = 95.95, c = 122.89 Å.
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Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of SET/TAF-Iβ ΔN from Homo sapiens
Wed, 07/28/2010 - 23:00The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iβ (TAF-Iβ), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-Iβ ΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 Å resolution and belonged to space group P43212.
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Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli
Wed, 07/28/2010 - 23:00Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4132, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å3 Da−1 and 51.77%, respectively, for two molecules in the asymmetric unit.
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Crystallization and preliminary crystallographic analysis of the ADP-ribosyltransferase HopU1
Wed, 07/28/2010 - 23:00Several 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.
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Crystallization and preliminary X-ray crystallographic studies of human FAIM protein
Wed, 07/28/2010 - 23:00Fas apoptosis inhibitory molecule (FAIM), an antagonist of Fas-induced cell death, is highly conserved and is broadly expressed in many tissues. It has been found that FAIM can stimulate neurite outgrowth in PC12 cells and primary neurons. However, the molecular mechanisms of action of FAIM are not understood in detail. Here, full-length human FAIM and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1–90) was crystallized and diffracted to a resolution of 2.5 Å; the crystal belonged to space group P31, with unit-cell parameters a = b = 58.02, c = 71.11 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit.
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Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase
Wed, 07/28/2010 - 23:00Recombinant 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.
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Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve
Wed, 07/28/2010 - 23:00The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution.
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Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar `switch complex' in Vibrio cholerae
Wed, 07/28/2010 - 23:00Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1–CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the `switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni–NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 Å3 Da−1 (47% solvent content) assuming the presence of one molecule per asymmetric unit.
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Expression, purification and crystallization of a plant type III polyketide synthase that produces diarylheptanoids
Wed, 07/28/2010 - 23:00Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase that catalyzes the one-pot formation of bisdemethoxycurcumin by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. Recombinant CUS was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 72.7, b = 97.2, c = 126.2 Å, α = γ = 90.0, β = 103.7°. A diffraction data set was collected in-house to 2.5 Å resolution.
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Crystallization and preliminary X-ray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coli
Wed, 07/28/2010 - 23:00Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts α-galactose to β-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected to a resolution of 1.9 Å from a single flash-cooled crystal that belonged to space group P212121.
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Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana
Wed, 07/28/2010 - 23:00Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the β-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.
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Crystallization and preliminary X-ray diffraction analysis of Pseudomonas aeruginosaphosphorylcholine phosphatase
Wed, 07/28/2010 - 23:00Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine to produce choline and inorganic phosphate. Phosphorylcholine is released by the action of haemolytic phospholipase C (PlcH) on phosphatidylcholine or sphingomyelin. PchP belongs to the HAD superfamily and its activity is dependent on Mg2+, Zn2+ or Cu2+. The possible importance of PchP in the pathogenesis of P. aeruginosa, the lack of information about its structure and its low identity to other members of this family led us to attempt its crystallization in order to solve its three-dimensional structure. Crystals of the protein have been grown and diffraction data have been obtained to 2.7 Å resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 137.16, b = 159.15, c = 73.31 Å, β = 117.89°. Statistical analysis of the unit-cell contents and the self-rotation function suggest a tetrameric state of the molecule with 222 point-group symmetry.
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Crystallization and preliminary X-ray analysis of the complex of human α-thrombin with a modified thrombin-binding aptamer
Wed, 07/28/2010 - 23:00The thrombin-binding aptamer (TBA) is a consensus DNA 15-mer that binds specifically to human α-thrombin at nanomolar concentrations and inhibits its procoagulant functions. Recently, a modified TBA (mTBA) containing a 5′–5′ inversion-of-polarity site has been shown to be more stable and to possess a higher thrombin affinity than its unmodified counterpart. The structure of the thrombin–TBA complex has previously been determined at low resolution, but did not provide a detailed picture of the aptamer conformation or of the protein–DNA assembly, while that of the complex with mTBA is unknown. Crystallographic analysis of the thrombin–mTBA complex has been attempted. The crystals diffracted to 2.15 Å resolution and belonged to space group I222.
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Crystallization of BMP receptor type IA bound to the antibody Fab fragment AbD1556
Wed, 07/28/2010 - 23:00An antibody Fab fragment, AbD1556, was selected against the extracellular domain of BMP receptor type IA, which blocks the binding of BMP-2 to BMPR-IA and thereby neutralizes BMP-2 activity. To study the mechanism by which BMPR-IA is recognized and bound by the Fab fragment, the complex of AbD1556 bound to BMPR-IA was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group P21, with unit-cell parameters a = 89.32, b = 129.25, c = 100.24 Å, β = 92.27°.
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Refined structures of placental alkaline phosphatase show a consistent pattern of interactions at the peripheral site
Mon, 07/26/2010 - 23:00In order to gain deeper insights into the functional sites of human placental alkaline phosphatase, the structures of the enzyme with the putative regulators l-Phe, pNPP and 5′-AMP [Llinas et al. (2005), J. Mol. Biol. 350, 441–451] were re-refined. Significant variations in ligand positioning and identity were found compared with the previous report. The multiple corrections to the model improved the phases and the electron-density maps, allowing the modeling of omitted side chains and multiple disordered residues. These improvements led to a change in the position of l-Phe at the peripheral binding site, which appeared to be reversed. The structure with pNPP contained only p-nitrophenol in three distinct sites, while the structure with 5′-AMP contained the p-nitrophenyl group in two of the sites instead of 5′-AMP. Comparison of the re-refined models shows a consistent pattern of interactions at the peripheral site.
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Structure of a fibronectin type III-like module from Clostridium thermocellum
Mon, 07/26/2010 - 23:00The 1.6 Å resolution structure of a fibronectin type III-like module from Clostridium thermocellum (PDB code 3mpc) with two molecules in the asymmetric unit is reported. The crystals used for data collection belonged to space group P212121, with unit-cell parameters a = 35.43, b = 45.73, c = 107.72 Å, and the structure was refined to an R factor of 0.166. Structural comparisons found over 800 similar structures in the Protein Data Bank. The broad range of different proteins or protein domains with high structural similarity makes it especially demanding to classify these proteins. Previous studies of fibronectin type III-like modules have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules or as proteins that help large enzyme complexes remain soluble.
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Structural studies of the catalytic core of the primate foamy virus (PFV-1) integrase
Mon, 07/26/2010 - 23:00Retroviral integrases are vital enzymes in the viral life cycle and thus are important targets for antiretroviral drugs. The structure of the catalytic core domain of the integrase from human foamy virus, which is related to HIV-1, has been solved. The structure of the protein is presented in two different crystal forms, each containing several molecules in the asymmetric unit, with and without the essential manganese or magnesium ion, and the structures are compared in detail. This allows regions of high structural variability to be pinpointed, as well as the effect of divalent cations on the conformation of the catalytic site.
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Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands
Mon, 07/26/2010 - 23:00Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in.
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