scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a ferredoxin/flavodoxin-NADP(H) oxidoreductase (Bc0385) fromBacillus cereus

2014 ◽  
Vol 70 (6) ◽  
pp. 777-780 ◽  
Author(s):  
Silje Skråmo ◽  
Hans-Petter Hersleth ◽  
Marta Hammerstad ◽  
K. Kristoffer Andersson ◽  
Åsmund K. Røhr
Keyword(s):  
Electron Transfer ◽  
Thioredoxin Reductase ◽  
Free Form ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Ferredoxin/flavodoxin-NADP(H) oxidoreductases (FNRs) are key enzymes involved in catalysing electron transfer between ferredoxins/flavodoxins and NAD(P)H/NAD(P)+. InBacillus cereusthere are three genes that may encode FNRs, and the Bc0385 FNR has been cloned, overexpressed, purified and successfully crystallized in its NADPH/NADP+-free form. Diffraction data have been collected to 2.5 Å resolution from crystals belonging to the orthorhombic space groupP21212, with unit-cell parametersa= 57.2,b= 164.3,c= 95.0 Å, containing two FNR molecules in the asymmetric unit. The structure of the Bc0385 FNR has been solved by molecular replacement, and is a member of the homodimeric thioredoxin reductase-like class of FNRs.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophileHalothermothrix orenii

2012 ◽  
Vol 68 (2) ◽  
pp. 240-243 ◽  
Author(s):  
Lokesh D. Kori ◽  
Andreas Hofmann ◽  
Bharat K. C. Patel
Keyword(s):  
Metal Ion ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Metal Ion Affinity Chromatography

A ribokinase gene (rbk) from the anaerobic halothermophilic bacteriumHalothermothrix oreniiwas cloned and overexpressed inEscherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 61.1,c= 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

1999 ◽  
Vol 55 (6) ◽  
pp. 1231-1233 ◽  
Author(s):  
Ivana Smatanová ◽  
Yuji Nagata ◽  
L. Anders Svensson ◽  
Masamichi Takagi ◽  
Jaromír Marek
Keyword(s):  
Diffusion Method ◽  
Sphingomonas Paucimobilis ◽  
Asymmetric Unit ◽  
Haloalkane Dehalogenase ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18–20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 8.9. The crystals diffract to at least 1.60 Å using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 Å. The asymmetric unit contains one molecule of the enzyme.

scholarly journals Expression, purification and preliminary crystallographic analysis ofMycobacterium tuberculosisCysQ, a phosphatase involved in sulfur metabolism

2014 ◽  
Vol 70 (6) ◽  
pp. 750-753 ◽  
Author(s):  
Anna I. Erickson ◽  
Reta D. Sarsam ◽  
Andrew J. Fisher
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Sulfur Metabolism ◽  
Crystallographic Analysis ◽  
Transfer Reactions ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

CysQ is part of the sulfur-activation pathway that dephosphorylates 3′-phosphoadenosine 5′-monophosphate (PAP) to regenerate adenosine 5′-monophosphate (AMP) and free phosphate. PAP is the product of sulfate-transfer reactions from sulfotransferases that use the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS). In some organisms PAP is also the product of PAPS reductases that reduce sulfate from PAPS to sulfite. CysQ fromMycobacterium tuberculosis, which plays an important role in the biosynthesis of sulfated glycoconjugates, was successfully purified and crystallized in 24% PEG 1500, 20% glycerol. X-ray diffraction data were collected to 1.7 Å resolution using a synchrotron-radiation source. Crystals grew in the orthorhombic space groupP212121, with unit-cell parametersa= 40.3,b= 57.9,c= 101.7 Å and with one monomer per asymmetric unit.

scholarly journals Purification, crystallization and preliminary X-ray analysis of the periplasmic haem-binding protein HutB fromVibrio cholerae

2015 ◽  
Vol 71 (4) ◽  
pp. 401-404 ◽  
Author(s):  
Shubhangi Agarwal ◽  
Maitree Biswas ◽  
Jhimli Dasgupta
Keyword(s):  
Pathogenic Bacteria ◽  
Molecular Level ◽  
Binding Protein ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Structural Insights

The mechanism of haem transport across the inner membrane of pathogenic bacteria is currently insufficiently understood at the molecular level and no information is available for this process inVibrio cholerae. To obtain structural insights into the periplasmic haem-binding protein HutB fromV. cholerae(VcHutB), which is involved in haem transport through the HutBCD haem-transport system, at the atomic level, VcHutB was cloned, overexpressed and crystallized using 1.6 Mammonium sulfate as a precipitant at pH 7.0. X-ray diffraction data were collected to 2.4 Å resolution on the RRCAT PX-BL-21 beamline at the Indus-2 synchrotron, Indore, India. The crystals belonged to space groupP43212, with unit-cell parametersa=b= 62.88,c= 135.8 Å. Matthews coefficient calculations indicated the presence of one monomer in the asymmetric unit, with an approximate solvent content of 45.02%. Molecular-replacement calculations withPhaserconfirmed the presence of a monomer in the asymmetric unit.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of theS-adenosylhomocysteine hydrolase (SAHH) fromThermotoga maritima

2014 ◽  
Vol 70 (11) ◽  
pp. 1563-1565 ◽  
Author(s):  
Miao He ◽  
Yingying Zheng ◽  
Chun-Hsiang Huang ◽  
Guojun Qian ◽  
Xiansha Xiao ◽  
...  
Keyword(s):  
Initial Phase ◽  
Thermotoga Maritima ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Adenosylhomocysteine Hydrolase ◽  
X Ray ◽  
Cell Parameters

S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion ofS-adenosylhomocysteine into adenosine and homocysteine. The SAHH fromThermotoga maritima(TmSAHH) was expressed inEscherichia coliand the recombinant protein was purified and crystallized.TmSAHH crystals belonging to space groupC2, with unit-cell parametersa= 106.3,b= 112.0,c= 164.9 Å, β = 103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85 Å resolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress.

scholarly journals Protein purification, crystallization and preliminary X-ray diffraction analysis ofL-arabinose isomerase fromLactobacillus fermentumCGMCC2921

2015 ◽  
Vol 71 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Zheng Xu ◽  
Sha Li ◽  
Jinfeng Liang ◽  
Xiaohai Feng ◽  
Hong Xu
Keyword(s):  
Monomethyl Ether ◽  
Size Exclusion ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Arabinose Isomerase

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose, as well as that of D-galactose to D-tagatose. A thermophilic AI derived fromLactobacillus fermentumCGMCC2921 (LFAI) was overexpressed inEscherichia coliBL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 Mbis-tris pH 6.5, 23% PEG 3350, 0.3 MNaCl (form 1 crystals) or 0.1 Mbis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 85.11,b= 184.57,c= 186.26 Å, α = β = γ = 90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da−1and a solvent content of 46.22%.

scholarly journals Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

2017 ◽  
Vol 73 (11) ◽  
pp. 607-611
Author(s):  
Fang Lu ◽  
Bei Zhang ◽  
Yong Liu ◽  
Ying Song ◽  
Gangxing Guo ◽  
...  
Keyword(s):  
Unit Cell ◽  
Diffusion Method ◽  
Data Sets ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

Phytases are phosphatases that hydrolyze phytates to less phosphorylatedmyo-inositol derivatives and inorganic phosphate. β-Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β-propeller phytase fromBacillussp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vapour-diffusion method in a condition consisting of 0.2 Msodium chloride, 0.1 MTris pH 8.5, 25%(w/v) PEG 3350 at 289 K. X-ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH-DI crystals belonged to space groupC121, with unit-cell parametersa = 156.84,b = 45.54,c = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH-DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1and a solvent content of 43.26%. Crystals of selenomethionine-substituted PhyH-DI belonged to space groupC2221, with unit-cell parametersa = 94.71,b= 97.03,c= 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1and a solvent content of 49.64%. Initial phases for PhyH-DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH-DI.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

Preparation and preliminary X-ray diffraction studies of a new crystal form of L-asparaginase from Escherichia coli

1999 ◽  
Vol 55 (9) ◽  
pp. 1616-1617
Author(s):  
I. Polikarpov ◽  
R. T. de Oliveira ◽  
J. Abrahão-Neto
Keyword(s):  
Escherichia Coli ◽  
Synchrotron Radiation ◽  
Crystal Form ◽  
Asymmetric Unit ◽  
Chemotherapeutic Drug ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Synchrotron Radiation Diffraction

L-Asparaginase is an enzyme which hydrolyzes asparagine to produce aspartic acid and ammonia. It is an effective chemotherapeutic drug, especially in the treatment of acute lymphoblastic leukaemia in children. The enzyme from Escherichia coli was crystallized in a new crystal form with space group C2, unit-cell parameters a = 76.3 (0), b = 134.6 (2), c = 64.8 (7) Å, β = 110.5 (1)° and a dimer in the asymmetric unit. Synchrotron-radiation diffraction data have been collected to 1.95 Å resolution.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of human DNA primase

2014 ◽  
Vol 70 (2) ◽  
pp. 206-210 ◽  
Author(s):  
Andrey G. Baranovskiy ◽  
Jianyou Gu ◽  
Nigar D. Babayeva ◽  
Vinod B. Agarkar ◽  
Yoshiaki Suwa ◽  
...  
Keyword(s):  
Active Site ◽  
Large Subunit ◽  
Structural Studies ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Human Dna ◽  
Structural Details

Human primase synthesizes RNA primers and transfers them to the active site of Pol α with subsequent extension with dNTPs. Human primase is a heterodimer of two subunits: a small catalytic subunit (p49) and a large subunit (p58). The structural details of the initiation and elongation steps of primer synthesis, as well as primer length counting, are not known. To address these questions, structural studies of human primase were initiated. Two types of crystals were obtained. The best diffracting crystals belonged to space groupP1, with unit-cell parametersa= 86.2,b= 88.9,c= 94.68 Å, α = 93.82, β = 96.57, γ = 111.72°, and contained two heterodimers of full-length p49 and p59 subunits in the asymmetric unit.

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