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Synchrotron-radiation Source (synchrotron-radiation + source)
Selected AbstractsCrystallization and preliminary crystallographic studies of MOMP (major outer membrane protein) from Campylobacter jejuniACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Jean Michel Bolla Campylobacter jejuni is the leading bacterial cause of human enteritis linked to ingestion of contaminated food or water. MOMP, the major outer membrane protein from these Gram-negative bacteria, belongs to the porin family. In order to determine the three-dimensional structure of this protein and to elucidate the underlying molecular mechanisms, the MOMP from C. jejuni strain 85H has been purified and crystallized by vapour diffusion. Two crystal forms were characterized for this membrane protein. X-ray diffraction data were collected to a resolution of 3.1,Å using a synchrotron-radiation source from the orthorhombic crystal form, which belonged to space group P21212 with unit-cell parameters a = 170.1, b = 101.9, c = 104.9,Å. With a trimer in the asymmetric unit, the solvent content is 64% (VM = 3.4,Å,Da,1). The other form exhibits trigonal symmetry (space group R3) with hexagonal unit-cell parameters a = b = 94.2, c = 161.2,Å, but diffracts X-rays poorly to about 4,Å with significant anisotropy. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Klebsiella pneumoniae maltohexaose-producing ,-amylaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Mitsuru Momma A recombinant form of Klebsiella pneumoniae maltohexaose-producing ,-amylase has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the microbatch technique using 80,mM sodium/potassium phosphate buffer pH 6.2 containing 8% polyethylene glycol 3000, 4% polyethylene glycol 3350 and 40,mM sodium thiocyanate. Crystals of the overexpressed recombinant enzyme diffracted to better than 2.5,Å resolution at 95,K using a synchrotron-radiation source. The crystals belong to the primitive monoclinic space group P21, with unit-cell parameters a = 74.8, b = 107.6, c = 82.2,Å, , = 96.2°. Assuming the presence of two molecules per asymmetric unit, the VM value for the crystal was 2.3,Å3,Da,1, indicating a solvent content of 47%. [source] Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cellsACTA CRYSTALLOGRAPHICA SECTION D, Issue 12-2 2004Toshihiko Akiba Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37,kDa inactive protein is proteolytically activated to a 30,kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P61 or P65, with unit-cell parameters a = b = 134.37, c = 121.24,Å. Diffraction data from a native crystal were collected to 2.75,Å resolution using a synchrotron-radiation source. [source] Expression, purification, crystallization and preliminary X-ray diffraction data of methylmalonate-semialdehyde dehydrogenase from Bacillus subtilisACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2004Hélène Dubourg Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Suitable crystals for X-ray diffraction experiments were obtained by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belong to space group P212121, with unit-cell parameters a = 195.2, b = 192.5, c = 83.5,Å, and contain one tetramer per asymmetric unit. X-ray diffraction data were collected to 2.5,Å resolution using a synchrotron-radiation source. The crystal structure was solved by the molecular-replacement method. [source] Expression, crystallization and preliminary X-ray crystallographic studies of Arthrobacter globiformis inulin fructotransferaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003Mitsuru Momma A recombinant form of Arthrobacter globiformis inulin fructotransferase (DFAIII-producing) has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293,K by the hanging-drop vapour-diffusion technique using 0.1,M Na HEPES pH 7.5 buffer containing 1.5,M lithium sulfate as a precipitant. Crystals of the recombinant wild-type enzyme diffracted to better than 1.5,Å at 100,K using a synchrotron-radiation source at the Photon Factory. The crystal belonged to space group R32, with unit-cell parameters a = b = 92.02, c = 229.82,Å in the hexagonal axes. Assuming the presence of one molecule in the asymmetric unit, the VM value for the crystal was 2.15,Å3,Da,1, indicating a solvent content of 42.8%. Selenomethionine-derivative crystals belonged to a different space group, C2, with unit-cell parameters a = 159.32, b = 91.92, c = 92.58,Å, , = 125.06. Matthews coefficient calculations suggested that the C2 selenomethionine-derivative crystal contained three molecules per asymmetric unit. [source] Crystallization and preliminary X-ray analysis of substrate complexes of leucine dehydrogenase from Thermoactinomyces intermediusACTA CRYSTALLOGRAPHICA SECTION D, Issue 6-2 2002Tatyana A. Muranova Leucine dehydrogenase is an octameric enzyme which belongs to the superfamily of amino-acid dehydrogenases and catalyses the reversible oxidative deamination of leucine to 2-ketoisocaproate, with the corresponding reduction of the cofactor NAD+. Catalysis by this enzyme is thought to involve a large-scale motion of the enzyme's two domains between an `open' and `closed' form, with the latter representing a conformation of the enzyme in which the partners involved in the hydride-transfer reaction are appropriately positioned for catalysis. Whilst a structure for the open form of the enzyme has been determined, the nature of the closed form has yet to be observed. In order to trap a closed form, crystals of the complexes of leucine dehydrogenase from Thermoactinomyces intermedius with 2-ketoisocaproate and with 2-ketoisocaproate and NAD+ have been obtained by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals of the binary complex with 2-ketoisocaproate belong to space group P212121, with approximate unit-cell parameters a = 106, b = 118, c = 320,Å and an octamer in the asymmetric unit, corresponding to a VM of 3.1,Å3,Da,1. The crystals of the non-productive ternary complex belong to space group P61 or P65, with approximate unit-cell parameters a = b = 117, c = 502,Å and an octamer in the asymmetric unit, corresponding to a VM of 3.0,Å3,Da,1. These crystals diffract X-rays on a synchrotron-radiation source to at least 2.8 and 3.3,Å resolution, respectively, and are suitable for a full structure determination. [source] Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasmaACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2001Ana L. Carvalho The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42,Å resolution were collected at the ESRF synchrotron-radiation source. [source] Crystallization and secondary-structure determination of a protein of the Lrp/AsnC family from a hyperthermophilic archaeonACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Norio Kudo A protein belonging to the Lrp/AsnC transcription-factor family, pot1216151, from the hyperthermophilic archaeon Pyrococcus sp. OT3 was crystallized. In Escherichia coli, leucine-responsive protein (Lrp) and AsnC regulate a number of metabolic genes. The crystals of pot1216151 diffracted to 2.3,Å using a conventional X-ray source and to 1.8,Å using a synchrotron-radiation source. The space group was identified to be P3121 or P3221, with unit-cell parameters a = b = 96.9, c = 98.5,Å. In combination with diffraction data obtained from K2[Pt(CN)6] and K(AuCl4) derivatives, an electron-density map was calculated at a resolution of 3.0,Å. Four monomers were identified in the asymmetric unit, with four ,-strands and two ,-helices in each monomer. [source] Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilusACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001Jacky Burke Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) is a 39.5,kDa molecular weight metalloenzyme which catalyzes the oxidation of glycerol to dihydroxyacetone with the concomitant reduction of NAD+ to NADH. Despite its classification as a member of the `iron-containing' polyol dehydrogenase family, studies on recombinant B. stearothermophilus GlyDH have shown this enzyme to be Zn2+ -dependent. Crystals of a S305C GlyDH mutant were obtained by the hanging-drop vapour-diffusion method, using ammonium sulfate and PEG 400 as precipitating agents, in the presence and absence of NAD+. The crystals belong to space group I422, with approximate unit-cell parameters a = b = 105, c = 149,Å and one subunit in the asymmetric unit, corresponding to a packing density of 2.6,Å3,Da,1. The crystals diffract X-rays to at least 1.8,Å resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinations of catalytic activity of this class of enzymes, for which no structures are currently available. [source] Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venomACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2000F. Canduri Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7,Å resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6222 (P6422). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation. [source] Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianumACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Francieli Colussi The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source. [source] Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2010Sachin Wakadkar Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4,M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9,Å resolution at 100,K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P61/P65, with unit-cell parameters a = 90.2, c = 314.5,Å and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure,activity relationship, assembly and thermal stability of Cu,ZnSODs. [source] Preliminary X-ray crystallographic analysis of the breakage,reunion domain of the GyrA subunit of DNA gyrase from Colwellia psychrerythraea strain 34HACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Ha Yun Jung DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal breakage,reunion domain of the GyrA subunit of DNA gyrase from Colwellia psychrerythraea 34H was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.60,Å resolution using a synchrotron-radiation source. The crystal belonged to space group P212121, with unit-cell parameters a = 98.98, b = 101.56, c = 141.83,Å. The asymmetric unit contained two molecules, with a corresponding VM of 3.18,Å3,Da,1 and a solvent content of 59.9%. [source] Purification and crystallization of the entire recombinant subunit E of the energy producer A1Ao ATP synthaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Asha Manikkoth Balakrishna A1Ao ATP synthases are the major energy producers in archaea. Subunit E of the stator domain of the ATP synthase from Pyrococcus horikoshii OT3 was cloned, expressed and purified to homogeneity. The monodispersed protein was crystallized by vapour diffusion. A complete diffraction data set was collected to 3.3,Å resolution with 99.4% completeness using a synchrotron-radiation source. The crystals belonged to space group I4, with unit-cell parameters a = 112.51, b = 112.51, c = 96.25,Å, and contained three molecules in the asymmetric unit. [source] Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thalianaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Jens Radzimanowski The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95,Å resolution using a synchrotron-radiation source and belonged to the hexagonal space group P61, with unit-cell parameters a = b = 96.1, c = 69.3,Å. [source] Crystallization and preliminary X-ray crystallographic analysis of DNA gyrase GyrB subunit from Xanthomonas oryzae pv. oryzaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Ha Yun Jung DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal fragment of the GyrB subunit of DNA gyrase from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.10,Å resolution using a synchrotron-radiation source. The crystal belonged to space group I41, with unit-cell parameters a = b = 110.27, c = 70.75,Å. The asymmetric unit contained one molecule, with a VM of 2.57,Å3,Da,1 and a solvent content of 50.2%. [source] Crystallization and X-ray diffraction data collection of topoisomerase IV ParE subunit from Xanthomonas oryzae pv. oryzaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Hye Jeong Shin Topoisomerase IV is involved in topological changes in the bacterial genome using the free energy from ATP hydrolysis. Its functions are the decatenation of daughter chromosomes following replication by DNA relaxation and double-strand DNA breakage. In this study, the N-terminal fragment of the topoisomerase IV ParE subunit from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.15,Å resolution using a synchrotron-radiation source. The crystal belonged to space group P42212, with unit-cell parameters a = b = 105.30, c = 133.76,Å. The asymmetric unit contains one molecule, with a corresponding VM of 4.21,Å3,Da,1 and a solvent content of 69.6%. [source] Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complexACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Carlos R. Ruiz-Martínez The native oxygen-carrier haemoglobins complex (HbII,III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20,Å, respectively, using a 0.886,Å synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P42212, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49,Å for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (VM) of 3.08 and 3.06,Å3,Da,1 for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume. [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] Crystallization and preliminary X-ray analysis of isomaltase from Saccharomyces cerevisiaeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2008Keizo Yamamoto Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77,Å, , = 91.17°. X-ray diffraction data were collected to 1.35,Å resolution from a single crystal on a synchrotron-radiation source. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of chlorite dismutase: a detoxifying enzyme producing molecular oxygenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008Daniël C. De Geus Chlorite dismutase, a homotetrameric haem-based protein, is one of the key enzymes of (per)chlorate-reducing bacteria. It is highly active (>2,kU,mg,1) in reducing the toxic compound chlorite to the innocuous chloride anion and molecular oxygen. Chlorite itself is produced as the intermediate product of (per)chlorate reduction. The chlorite dismutase gene in Azospira oryzae strain GR-1 employing degenerate primers has been identified and the active enzyme was subsequently overexpressed in Escherichia coli. Chlorite dismutase was purified, proven to be active and crystallized using sitting drops with PEG 2000 MME, KSCN and ammonium sulfate as precipitants. The crystals belonged to space group P21212 and were most likely to contain six subunits in the asymmetric unit. The refined unit-cell parameters were a = 164.46, b = 169.34, c = 60.79,Å. The crystals diffracted X-rays to 2.1,Å resolution on a synchrotron-radiation source and a three-wavelength MAD data set has been collected. Determination of the chlorite dismutase structure will provide insights into the active site of the enzyme, for which no structures are currently available. [source] Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotBACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2008Anna Roujeinikova The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P21, with unit-cell parameters a = 50.8, b = 89.5, c = 66.3,Å, , = 112.5°. The crystals diffract X-rays to at least 1.6,Å resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38,Å showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data. [source] Crystallization and preliminary X-ray data analysis of ,-alanine synthase from Drosophila melanogasterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2007Stina Lundgren ,-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3,Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3,Å, , = 125.8°) and contain 8,10 molecules per asymmetric unit. [source] The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondriaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2006Jocelyn A. Silvester A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79,184 of subunit b, residues 1,124 of subunit d and the entire F6 subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8,Å resolution. They belong to the monoclinic space group P21. [source] Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37RvACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2006Ana Luiza Vivan Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I212121, with unit-cell parameters a = 98.26, b = 133.22, c = 225.01,Å, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2,Å resolution using a synchrotron-radiation source. [source] Crystallization and preliminary X-ray analysis of PH1566, a putative ribosomal RNA-processing factor from the hyperthermophilic archaeon Pyrococcus horikoshii OT3ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006Min Ze Jia A putative ribosomal RNA-processing factor consisting of two KH domains from Pyrococcus horikoshii OT3 (PH1566; 25,kDa) was crystallized by the sitting-drop vapour-diffusion method using PEG 3000 as the precipitant. The crystals diffracted X-rays to beyond 2.0,Å resolution using a synchrotron-radiation source. The space group of the crystals was determined as primitive orthorhombic P212121, with unit-cell parameters a = 45.9, b = 47.4, c = 95.7,Å. The crystals contain one molecule in the asymmetric unit (VM = 2.5,Å3,Da,1) and have a solvent content of 50%. [source] Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2005Elizabeth Pleshe Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2,-hydroxyl group of the ribose sugar, which is performed by a number of S -adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the ,/,-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85,Å resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the ,/,-knot superfamily, with ,-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding. [source] Expression, purification and X-ray crystallographic analysis of thioredoxin from Streptomyces coelicolorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2005Petra Stefankova Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. In turn, they are reduced by electrons obtained from the NADPH-containing thioredoxin reductase. Thioredoxins have been isolated and characterized from a large number of organisms. The Gram-positive bacterium Streptomyces coelicolor contains three thioredoxins that are involved in unknown biological processes. trxA from S. coelicolor was cloned and expressed in Escherichia coli and the protein purified and crystallized using the hanging-drop method of vapour diffusion. The crystal structure of thioredoxin A has been determined at 1.5,Å resolution using a synchrotron-radiation source. The protein reveals a thioredoxin-like fold with a typical CXXC active site. The crystal exhibits the symmetry of space group P21212, with unit-cell parameters a = 43.6, b = 71.8, c = 33.2,Å. [source] Application of protein engineering to enhance crystallizability and improve crystal propertiesACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Zygmunt S. Derewenda Until recently, protein crystallization has mostly been regarded as a stochastic event over which the investigator has little or no control. With the dramatic technological advances in synchrotron-radiation sources and detectors and the equally impressive progress in crystallographic software, including automated model building and validation, crystallization has increasingly become the rate-limiting step in X-ray diffraction studies of macromolecules. However, with the advent of recombinant methods it has also become possible to engineer target proteins and their complexes for higher propensity to form crystals with desirable X-ray diffraction qualities. As most proteins that are under investigation today are obtained by heterologous overexpression, these techniques hold the promise of becoming routine tools with the potential to transform classical crystallization screening into a more rational high-success-rate approach. This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application. [source] | ||||||||||