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Monoclinic Space Group C2 (monoclinic + space_group_c2)
Selected AbstractsStructure of the dodecamer r(GAUCACUUCGGU) with four 5,-overhang nucleotidesACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2004r(GAUCACUUCGGU) The crystal structure of an RNA dodecamer, r(GAUCACUUCGGU), was solved at 2.6,Å resolution by the molecular-replacement method and refined to an Rwork of 18.8% (Rfree = 22.8%) using 2494 reflections. The dodecamer crystallized in the monoclinic space group C2, with unit-cell parameters a = 71.34, b = 39.98, c = 32.47,Å, , = 104.7° and two independent strands in the asymmetric unit. The dodecamer adopts an octamer duplex structure with four 5,-overhang residues (G1A2U3C4), which form Watson,Crick base pairs with another four 5,-overhang residues of a symmetry-related duplex. The octamer duplex (ACUUCGGU) contains at its center four mismatched base pairs flanked by two Watson,Crick base pairs. The mismatched bases form two G·U wobble base pairs at the ends and two U·C base pairs at the center, with one base,base hydrogen bond N4(C)O4(U) and a water bridge connecting the N(3) of the cytosine and uridine. The present study reinforces the concept of the stability of the conformation of UUCG in RNA double-helical structures. [source] Crystallization and preliminary X-ray analysis of a thermoalkalophilic lipase from Bacillus stearothermophilus L1ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001Seong-Tae Jeong A thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) was crystallized in two different crystal forms using a low concentration of the enzyme and a calcium-exchange process. The first, needle-like, crystal form, which diffracts to about 3.5,Å, belongs to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 72.96, c = 104.41,Å. The second, monoclinic, crystal form, which behaves better than the first form for crystallographic analyses, belongs to the monoclinic space group C2 and has unit-cell parameters a = 119.62, b = 85.05, c = 98.36,Å, , = 99.73°. From the monoclinic crystals, a native data set and a samarium-derivative data set were collected to 2.0 and 2.3,Å resolution, respectively. The difference Patterson map between the two data sets shows strong heavy-atom peaks, indicating that the crystals are suitable for a high-resolution structure determination. [source] Crystallization and preliminary X-ray crystallographic studies of recombinant human betaine,homocysteine S-methyltransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Nandita Bose Betaine,homocysteine S-methyltransferase (BHMT) catalyzes a reaction essential for regulation of methionine and homocysteine metabolism and the catabolism of choline in mammalian tissues. Human recombinant BHMT (MW = 45,kDa) has been crystallized by the hanging-drop vapor-diffusion method at 294,K using ethylene glycol as the precipitant. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 109.190, b = 91.319, c = 88.661,Å, , = 122.044°, and diffract to 2.9,Å resolution on a local rotating-anode X-ray source. Rotation-function analysis and the Matthews coefficient, VM = 2.46,Å3,Da,1, are consistent with a dimer in the asymmetric unit, suggesting that the active enzyme is a tetramer with 222 symmetry. [source] Crystallization and preliminary X-ray analysis of the matrix protein from Ebola virusACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Andréa Dessen The matrix protein from Ebola virus is a membrane-associated molecule that plays a role in viral budding. Despite its functional similarity to other viral matrix proteins, it displays no sequence similarity and hence may have a distinct fold. X-ray diffraction quality crystals of the Ebola VP40 matrix protein were grown by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 64.4, b = 91.1, c = 47.9,Å, , = 96.3°. A data set to 1.9,Å resolution has been collected using synchrotron radiation. The unit cell contains one molecule of molecular weight 35,kDa per asymmetric unit, with a corresponding volume solvent content of 35%. [source] Crystallization and preliminary X-ray diffraction studies of d(ACGTAGCTACGT)2:[actinomycin D, (echinomycin)2] and d(ACGTAGCTACGT)2:[actinomycin D, (triostin A)2] complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2000Hana L. Takusagawa A DNA,multiple drug complex, d(ACGTAGCTACGT)2:[actinomycin D, (echinomycin)2] has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 85.6, b = 72.8, c = 56.6,Å, , = 101.5° at 93,K and Z = 8.,The crystal diffracted to 3.0,Å resolution along the DNA fiber axis and to 3.5,Å resolution in other directions. The Patterson maps indicate that all complexes in the crystal are oriented along their helical axes in the [10] direction. [source] Crystallization and preliminary X-ray crystallographic analysis of the ArsM arsenic(III) S -adenosylmethionine methyltransferaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Kavitha Marapakala Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III) S -adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 84.85, b = 46.89, c = 100.35,Å, , = 114.25° and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76,Å. [source] Crystallization and preliminary X-ray analysis of the major peanut allergen Ara,h,1 core regionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Cerrone Cabanos Peanuts contain some of the most potent food allergens known to date. Ara,h,1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara,h,1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1,M sodium citrate pH 5.6, 0.1,M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25,Å resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a = 156.521, b = 88.991, c = 158.971,Å, , = 107.144°. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan). [source] Crystallization and preliminary X-ray diffraction analysis of Pseudomonas aeruginosa phosphorylcholine phosphataseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010Lisandro H. Otero Pseudomonas 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, , = 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. [source] Structure of the newly found green turtle egg-white ribonucleaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Somporn Katekaew Marine green turtle (Chelonia mydas) egg-white ribonuclease (GTRNase) was crystallized from 1.1,M ammonium sulfate pH 5.5 and 30% glycerol using the sitting-drop vapour-diffusion method. The structure of GTRNase has been solved at 1.60,Å resolution by the molecular-replacement technique using a model based on the structure of RNase 5 (murine angiogenin) from Mus musculus (46% identity). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 86.271, b = 34.174, c = 39.738,Å, , = 90, , = 102, , = 90°. GTRNase consists of three helices and seven ,-strands and displays the ,+, folding topology typical of a member of the RNase A superfamily. Superposition of the C, coordinates of GTRNase and RNase A superfamily members indicates that the overall structure is highly similar to that of angiogenin or RNase 5 from M. musculus (PDB code 2bwl) and RNase A from Bos taurus (PDB code 2blz), with root-mean-square deviations of 3.9 and 2.0,Å, respectively. The catalytic residues are conserved with respect to the RNase A superfamily. The three disulfide bridges observed in the reptilian enzymes are conserved in GTRNase, while one further disulfide bond is required for the structural stability of mammalian RNases. GTRNase is expressed in egg white and the fact that its sequence has the highest similarity to that of snapping turtle pancreatic RNase suggests that the GTRNase secreted from oviduct cells to form egg white is probably the product of the same gene as activated in pancreatic cells. [source] Crystallization and preliminary X-ray crystallographic analysis of BxlA, an intracellular ,- d -xylosidase from Streptomyces thermoviolaceus OPC-520ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Hideaki Morioka BxlA from Streptomyces thermoviolaceus OPC-520, together with the extracellular BxlE and the integral membrane proteins BxlF and BxlG, constitutes a xylanolytic system that participates in the intracellular transport of xylan-degradation products and the production of xylose. To elucidate the mechanism of the hydrolytic degradation of xylooligosaccharides to xylose at the atomic level, X-ray structural analysis of BxlA was attempted. The recombinant BxlA protein (molecular weight 82,kDa) was crystallized by the hanging-drop vapour-diffusion method at 289,K. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 142.2, b = 129.5, c = 101.4,Å, , = 119.8°, and contained two molecules per asymmetric unit (VM = 2.47,Å3,Da,1). Diffraction data were collected to a resolution to 2.50,Å and provided a data set with an overall Rmerge of 8.3%. [source] Crystallization and preliminary X-ray analysis of Na-SAA-2 from the human hookworm parasite Necator americanusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Oluwatoyin A. Asojo Human hookworms are among the most pathogenic soil-transmitted helminths. These parasitic nematodes have co-evolved with the host and are able to maintain a high worm burden for decades without killing the human host. However, it is possible to develop vaccines against laboratory-challenge hookworm infections using either irradiated third-state infective larvae (L3) or enzymes from the adult parasites. In an effort to control hookworm infection globally, the Human Hookworm Vaccine Initiative, a product-development partnership with the Sabin Vaccine Institute to develop new control tools including vaccines, has identified a battery of protein antigens, including surface-associated antigens (SAAs) from L3. SAA proteins are characterized by a 13,kDa conserved domain of unknown function. SAA proteins are found on the surface of infective L3 stages (and some adult stages) of different nematode parasites, suggesting that they may play important roles in these organisms. The atomic structures and function of SAA proteins remain undetermined and in an effort to remedy this situation recombinant Na-SAA-2 from the most prevalent human hookworm parasite Necator americanus has been expressed, purified and crystallized. Useful X-ray data have been collected to 2.3,Å resolution from a crystal that belonged to the monoclinic space group C2 with unit-cell parameters a = 73.88, b = 35.58, c = 42.75,Å, , = 116.1°. [source] Crystallization and preliminary X-ray diffraction data analysis of stenodactylin, a highly toxic type 2 ribosome-inactivating protein from Adenia stenodactylaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Giovanna Tosi Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15,Å). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit. [source] Crystallization and preliminary X-ray diffraction studies of the carbohydrate-recognition domain of SIGN-R1, a receptor for microbial polysaccharides and sialylated antibody on splenic marginal zone macrophagesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Noella Silva-Martin SIGN-R1, or CD209b, is a mouse C-type lectin receptor that is expressed at high levels on macrophages in lymphoid tissues, especially within the marginal zone of the spleen. SIGN-R1 can bind and mediate the uptake of various microbial polysaccharides, including dextrans, lipopolysaccharides and pneumococcal capsular polysaccharides. It has been shown that SIGN-R1 mediates the clearance of encapsulated pneumococcus, complement fixation via binding C1q independent of antibody and innate resistance to pneumococcal infection. Recently, SIGN-R1 has also been demonstrated to bind sialylated antibody and mediate its activity to suppress autoimmunity. The carbohydrate-recognition domain (CRD) of SIGN-R1 has been cloned and overexpressed in a soluble secretory form in mammalian Chinese hamster ovary (CHO) cells. The CRD protein of SIGN-R1 was purified from CHO cell-culture supernatant and concentrated for crystallization using the hanging-drop vapour-diffusion method at 291,K. Crystals grew from a mixture of 2,M ammonium sulfate in 0.1,M bis-tris pH 5.5. Single crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 146.72, b = 92.77, c = 77.06,Å, , = 121.66°, allowed the collection of a full X-ray data set to a maximum resolution of 1.87,Å. [source] Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Saeyoung Lee Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O -3,6-anhydro-,- l -galactopyranosyl-(1,3)- d -galactose] using various ,-agarases. Neoagarobiose hydrolase is an enzyme that acts on the ,-1,3 linkage in neoagarobiose to yield d -galactose and 3,6-anhydro- l -galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11,Å, , = 101.86°. The crystals diffracted to 1.98,Å resolution and possibly contains two molecules in the asymmetric unit. [source] Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogasterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Yumiko Mishima Gram-negative bacteria-binding protein 3 (GNBP3) is a pattern-recognition receptor which contributes to the defensive response against fungal infection in Drosophila. The protein consists of an N-terminal domain, which is considered to recognize ,-glucans from the fungal cell wall, and a C-terminal domain, which is homologous to bacterial glucanases but devoid of activity. The N-terminal domain of GNBP3 (GNBP3-Nter) was successfully purified after expression in Drosophila S2 cells. Diffraction-quality crystals were produced by the hanging-drop vapour-diffusion method using PEG 2000 and PEG 8000 as precipitants. Preliminary X-ray diffraction analysis revealed that the GNBP3-Nter crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 134.79, b = 30.55, c = 51.73,Å, , = 107.4°, and diffracted to 1.7,Å using synchrotron radiation. The asymmetric unit is expected to contain two copies of GNBP3-Nter. Heavy-atom derivative data were collected and a samarium derivative showed one high-occupancy site per molecule. [source] Crystallization and preliminary X-ray analysis of 4-pyridoxolactonase from Mesorhizobium lotiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Sayoko Matsuda 4-Pyridoxolactonase from Mesorhizobium loti MAFF303099 has been overexpressed in Escherichia coli. The recombinant enzyme was purified and was crystallized by the sitting-drop vapour-diffusion method using PEG 4000 and ammonium sulfate as precipitants. Crystals of the free enzyme (form I) and of the 5-pyridoxolactone-bound enzyme (form II) grew under these conditions. Crystals of form I diffracted to 2.0,Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 77.93, b = 38.88, c = 81.60,Å, , = 117.33°. Crystals of form II diffracted to 1.9,Å resolution and belonged to the monoclinic space group C2, with unit-cell parameters a = 86.24, b = 39.35, c = 82.68,Å, , = 118.02°. The calculated VM values suggested that the asymmetric unit contains one molecule in both crystal forms. [source] Crystallization and preliminary X-ray analysis of AAMS amidohydrolase, the final enzyme in degradation pathway I of pyridoxineACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Jun Kobayashi ,-(N -Acetylaminomethylene)succinic acid (AAMS) amidohydrolase from Mesorhizobium loti MAFF303099, which is involved in a degradation pathway of vitamin B6 and catalyzes the degradation of AAMS to acetic acid, ammonia, carbon dioxide and succinic semialdehyde, has been overexpressed in Escherichia coli. To elucidate the reaction mechanism based on the tertiary structure, the recombinant enzyme was purified and crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as precipitant. A crystal of the enzyme belonged to the monoclinic space group C2, with unit-cell parameters a = 393.2, b = 58.3, c = 98.9,Å, , = 103.4°, and diffraction data were collected to 2.7,Å resolution. The VM value and calculation of the self-rotation function suggested that three dimers with a threefold symmetry were possibly present in the asymmetric unit. [source] Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Xenia Bogdanovi Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P212121) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9,Å, , = 109.3° for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7,Å, , = 112.4° for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6,Å. The crystals of the two different mutants diffracted X-rays beyond 2.0,Å resolution. [source] Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosineACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Henrik Ingvarsson Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni2+ -affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1,Å resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3,Å, , = 124.8°. The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1,Å3,Da,1. Structure determination is in progress. [source] Crystallization and preliminary X-ray analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Tatyana Prudnikova A novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P212121, while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2,Å resolution for both DbeA and DbeA1 crystals. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of YvoA from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Marcus Resch The putative transcriptional regulator protein YvoA (BSU35030) from Bacillus subtilis was cloned and heterologously expressed in Escherichia coli. The protein was purified by immobilized metal-affinity chromatography and size-exclusion chromatography and subsequently crystallized. A complete native data set was collected to 2.50,Å resolution. The crystals belonged to the monoclinic space group C2 and preliminary analysis of the diffraction data indicated the presence of approximately 12 molecules per asymmetric unit. [source] Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Louise Aigrain NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures. [source] Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seedsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Tales Rocha Moura Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293,K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1,M HEPES pH 7.5 and 3.0,M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99,Å, , = 90.0, , = 120.8, , = 90.0°. Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5,Å resolution. [source] Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Yong-Fu Li Enterotoxigenic Escherichia coli (ETEC), a major global cause of diarrhea, initiates the pathogenic process via fimbriae-mediated attachment to the small intestinal epithelium. A common prototypic ETEC fimbria, colonization factor antigen I (CFA/I), consists of a tip-localized minor adhesive subunit CfaE and the stalk-forming major subunit CfaB, both of which are necessary for fimbrial assembly. To elucidate the structure of CFA/I at atomic resolution, three recombinant proteins were generated consisting of fusions of the minor and major subunits (CfaEB) and of two (CfaBB) and three (CfaBBB) repeats of the major subunit. Crystals of CfaEB diffracted X-rays to 2.1,Å resolution and displayed the symmetry of space group P21. CfaBB exhibited a crystal diffraction limit of 2.3,Å resolution and had the symmetry of space group P21212. CfaBBB crystallized in the monoclinic space group C2 and diffracted X-rays to 2.3,Å resolution. These structures were determined using the molecular-replacement method. [source] Crystallization and preliminary X-ray crystallographic studies of the Z-DNA-binding domain of a PKR-like kinase (PKZ) in complex with Z-DNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Doyoun Kim PKZ, a PKR-like eIF2, kinase, consists of a Z-DNA-specific binding domain (Z,) and an eIF2, kinase domain. The kinase activity of PKZ is modulated by the binding of Z, to Z-DNA. The mechanisms underlying Z-DNA binding and the subsequent stimulation of PKZ raise intriguing questions. Interestingly, the Z-DNA-binding domain of PKZ from goldfish (Carassius auratus; caZ,PKZ) shows limited sequence homology to other canonical Z, domains, suggesting that it may have a distinct Z-DNA-recognition mode. In this study, the Z-DNA-binding activity and stoichiometry of caZ,PKZ were confirmed using circular dichroism (CD). In addition, preliminary X-ray studies of the caZ,PKZ,Z-DNA complex are reported as the first step in the determination of its three-dimensional structure. Bacterially expressed recombinant caZ,PKZ was purified and crystallized with Z-DNA at 295,K using the microbatch method. X-ray diffraction data were collected to 1.7,Å resolution with an Rmerge of 7.4%. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 55.54, b = 49.93, c = 29.44,Å, , = 96.5°. Structural analysis of caZ,PKZ,Z-DNA will reveal the binding mode of caZ,PKZ to Z-DNA and its relevance to other Z-DNA-binding proteins. [source] Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Thanh Thi Ngoc Doan The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNAGlu. It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8,Å resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1,Å, , = 96.3°. The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (VM) range of 2.70,2.02,Å3,Da,1 and a solvent-content range of 54.5,39.3%. [source] X-ray diffraction analysis of a human tRNAGly acceptor-stem microhelix isoacceptor at 1.18,Å resolutionACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009André Eichert Interest has been focused on comparative X-ray structure analyses of different tRNAGly acceptor-stem helices. tRNAGly/glycyl-tRNA synthetase belongs to the so-called class II system, in which the tRNA identity elements consist of simple and unique determinants that are located in the tRNA acceptor stem and the discriminator base. Comparative structure investigations of tRNAGly microhelices provide insight into the role of tRNA identity elements. Predominant differences in the structures of glycyl-tRNA synthetases and in the tRNA identity elements between prokaryotes and eukaryotes point to divergence during the evolutionary process. Here, the crystallization and high-resolution X-ray diffraction analysis of a human tRNAGly acceptor-stem microhelix with sequence 5,-G1C2A3U4U5G6G7 -3,, 5,-C66C67A68A69U70G71C72 -3, is reported. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 37.32, b = 37.61, c = 30.47,Å, , = 112.60° and one molecule per asymmetric unit. A data set was collected using synchrotron radiation and data were processed within the resolution range 50.0,1.18,Å. The structure was solved by molecular replacement. [source] Crystallization and preliminary X-ray crystallographic studies of the ,-class glutathione S -transferase from the Antarctic clam Laternula ellipticaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2008Eun Hyuk Jang Glutathione S -transferases are involved in phase II detoxification processes and catalyze the nucleophilic attack of the tripeptide glutathione on a wide range of endobiotic and xenobiotic electrophilic substrates. The ,-class glutathione S -transferase from Laternula elliptica was overexpressed in Escherichia coli, purified and crystallized with two substrates: glutathione and 1-chloro-2,4-dinitrobenzene (CDNB). Diffraction data were collected to 2.20,Å resolution for the glutathione-complex crystals and to 2.00,Å resolution for the CDNB-complex crystals using a synchrotron-radiation source. Both crystals belonged to the C -centred monoclinic space group C2. The unit-cell parameters for the CDNB-complex crystals were a = 89.66, b = 59.27, c = 55.45,Å, , = 124.52°. The asymmetric unit contained one molecule, with a corresponding VM of 2.36,Å3,Da,1 and a solvent content of 47.8%. [source] Expression, purification, crystallization and preliminary X-ray crystallographic studies of a novel acetylcitrulline deacetylase from Xanthomonas campestrisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2005Dashuang Shi A novel N -acetyl- l -citrulline deacetylase that is able to catalyze the hydrolysis of N -acetyl- l -citrulline to acetate and citrulline was identified from Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the monoclinic space group C2 and diffract to 1.75,Å resolution, with unit-cell parameters a = 94.13, b = 95.23, c = 43.61,Å, , = 93.76°. Since attempts to use homologous structural models to solve the structure via molecular replacement were unsuccessful, the selenomethionine-substituted protein was prepared using an overnight auto-induction overexpression system. Selenomethionine incorporation into the protein was verified by MALDI,TOF/TOF mass-spectroscopic analysis after trypsin digestion. The crystals of the selenomethionine-substituted protein were prepared using crystallization conditions similar to those for the native protein. Multiple anomalous dispersion (MAD) data were collected at Brookhaven National Laboratory. Structure determination is under way using the MAD phasing method. [source] Crystallization and preliminary X-ray analysis of coagulation factor IX-binding protein from habu snake venom at pH 6.5 and 4.6ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2005Nobuhiro Suzuki Coagulation factor IX-binding protein isolated from Trimeresurus flavoviridis (IX-bp) is a C-type lectin-like protein. It is an anticoagulant protein consisting of homologous subunits A and B. The subunits both contain a Ca2+ -binding site with differing affinity (Kd values of 14 and 130,µM at pH 7.5). These binding characteristics are pH-dependent; under acidic conditions, the affinity of the low-affinity site was reduced considerably. In order to identify which site has high affinity and also to investigate the Ca2+ -releasing mechanism, IX-bp was crystallized at pH 6.5 and 4.6. The crystals at pH 6.5 and 4.6 diffracted to 1.72 and 2.29,Å resolution, respectively; the former crystals belong to the monoclinic space group P21, with unit-cell parameters a = 60.7, b = 63.5, c = 66.9,Å, , = 117.0°, while the latter belong to the monoclinic space group C2, with a = 134.1, b = 37.8, c = 55.8,Å, , = 110.4°. [source] | ||||||||||