scholarly journals Purification and crystallographic analysis of a FAD-dependent halogenase fromStreptomycessp. JCM9888

2015 ◽  
Vol 71 (8) ◽  
pp. 972-976 ◽  
Author(s):  
Yanqun Zhao ◽  
Baohua Yan ◽  
Ting Yang ◽  
Jian Jiang ◽  
Heng Wei ◽  
...  
Keyword(s):  
Model Building ◽  
Structure Refinement ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Hexahistidine Tag ◽  
Crystallization Experiments

A new FAD (flavin adenine dinucleotide)-dependent halogenase HalY fromStreptomycessp. JCM9888 was reported to be involved in the regioselective halogenation of adenine. HalY is a variant B FAD-dependent halogenase that is most similar to the halogenase PltA involved in pyoluteorin biosynthesis. This study reports the overexpression and purification of HalY with an N-terminal hexahistidine tag, followed by crystallization experiments and X-ray crystallographic analysis. HalY was purified as a monomer in solution and crystallized to give X-ray diffraction to a resolution of 1.7 Å. The crystal belonged to the monoclinic space groupP21, with unit-cell parametersa= 41.4,b= 113.4,c= 47.6 Å, α = γ = 90, β = 107.4°, and contained one monomer of HalY in the asymmetric unit, with a calculated Matthews coefficient of 2.3 Å3 Da−1and a solvent content of 46%. The structure of the halogenase CndH was used as a search model in molecular replacement to obtain the initial model of HalY. Manual model building and structure refinement of HalY are in progress.

scholarly journals A putative siderophore-interacting protein from the marine bacteriumShewanella frigidimarinaNCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

2016 ◽  
Vol 72 (9) ◽  
pp. 667-671 ◽  
Author(s):  
Inês B. Trindade ◽  
Bruno M. Fonseca ◽  
Pedro M. Matias ◽  
Ricardo O. Louro ◽  
Elin Moe
Keyword(s):  
Iron Acquisition ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Interacting Protein ◽  
X Ray ◽  
Cell Parameters ◽  
Unit Structure ◽  
Shewanella Frigidimarina

Siderophore-binding proteins (SIPs) perform a key role in iron acquisition in multiple organisms. In the genome of the marine bacteriumShewanella frigidimarinaNCIMB 400, the gene tagged as SFRI_RS12295 encodes a protein from this family. Here, the cloning, expression, purification and crystallization of this protein are reported, together with its preliminary X-ray crystallographic analysis to 1.35 Å resolution. The SIP crystals belonged to the monoclinic space groupP21, with unit-cell parametersa= 48.04,b= 78.31,c= 67.71 Å, α = 90, β = 99.94, γ = 90°, and are predicted to contain two molecules per asymmetric unit. Structure determination by molecular replacement and the use of previously determined ∼2 Å resolution SIP structures with ∼30% sequence identity as templates are ongoing.

Crystallization and Preliminary X-ray Diffraction Analysis of Cold-Active β-Galactosidase from Arthrobacter sp. C2-2

2005 ◽  
Vol 70 (1) ◽  
pp. 124-132 ◽  
Author(s):  
Hana Petroková ◽  
Eva Vondráčková ◽  
Tereza Skálová ◽  
Jan Dohnálek ◽  
Petra Lipovová ◽  
...  
Keyword(s):  
Structure Refinement ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Phase Problem ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Psychrotrophic Bacteria ◽  
Cold Active

β-Galactosidase from psychrotrophic bacteria strainArthrobactersp. C2-2 catalyzes cleavage of β-D-galactosyl moieties from β-D-galactosides and is interesting for its activity at low temperatures. Various types of crystals with dimensions of up to 0.8 mm were obtained and X-ray diffraction data up to 1.9 Å were collected. The crystals belong to the monoclinic space groupP21with unit-cell parametersa= 140.1 Å,b= 205.7 Å,c= 140.5 Å and β = 102.3°. The enzyme (molecular weight of a monomer is 111 kDa) forms hexamers in the crystal structure (one hexamer per asymmetric unit). The phase problem was solved by molecular replacement. Structure refinement is in progress.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase fromAspergillus niger

2014 ◽  
Vol 70 (11) ◽  
pp. 1508-1512 ◽  
Author(s):  
Prem Prakash ◽  
Adhish S. Walvekar ◽  
Narayan S. Punekar ◽  
Prasenjit Bhaumik
Keyword(s):  
Glutamate Dehydrogenase ◽  
Model Building ◽  
Specific Activity ◽  
Structure Refinement ◽  
High Specific Activity ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Carbon And Nitrogen

Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study,Aspergillus nigerNADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Å resolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space groupR32, with unit-cell parametersa=b= 173.8,c= 241.5 Å, α = β = 90, γ = 120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of (S)-3-hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum

2014 ◽  
Vol 70 (4) ◽  
pp. 485-488
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Crystal Volume

(S)-3-Hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum(CbHBD) is an enzyme that catalyzes the second step in the biosynthesis ofn-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. TheCbHBD protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 2 Mammonium sulfate, 0.1 MCAPS pH 10.5, 0.2 Mlithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space groupR3, with unit-cell parametersa=b= 148.5,c= 201.6 Å. With four molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 3.52 Å3 Da−1, which corresponds to a solvent content of approximately 65.04%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Purification and X-ray crystallographic analysis of 7-keto-8-aminopelargonic acid (KAPA) synthase fromMycobacterium smegmatis

2014 ◽  
Vol 70 (10) ◽  
pp. 1372-1375 ◽  
Author(s):  
Shanghua Fan ◽  
Defeng Li ◽  
Joy Fleming ◽  
Yuan Hong ◽  
Tao Chen ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Unit Cell ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Essential Enzyme ◽  
Mycobacterial Growth

7-Keto-8-aminopelargonic acid synthase (KAPA synthase; BioF) is an essential enzyme for mycobacterial growth that catalyses the first committed step in the biotin-synthesis pathway. It is a pyridoxal 5′-phosphate (PLP)-dependent enzyme and is a potential drug target. Here, the cloning, expression, purification and crystallization of KAPA synthase fromMycobacterium smegmatis(MsBioF) and the characterization of MsBioF crystals using X-ray diffraction are described. The crystals diffracted to 2.3 Å resolution and belonged to the monoclinic space groupP21, with unit-cell parametersa= 70.88,b= 91.68,c= 109.84 Å, β = 97.8°. According to the molecular weight of MsBioF, the unit-cell parameters and the self-rotation function map, four molecules are present in each asymmetric unit with aVMvalue of 2.06 Å3 Da−1and a solvent content of 40.20%.

The new mineral novograblenovite, (NH4,K)MgCl3·6H2O from the Tolbachik volcano, Kamchatka, Russia: mineral description and crystal structure

2018 ◽  
Vol 83 (02) ◽  
pp. 223-231 ◽  
Author(s):  
Viktor M. Okrugin ◽  
Sharapat S. Kudaeva ◽  
Oxana V. Karimova ◽  
Olga V. Yakubovich ◽  
Dmitry I. Belakovskiy ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structure Refinement ◽  
Kamchatka Peninsula ◽  
New Mineral ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Tolbachik Volcano ◽  
Difference Fourier

AbstractThe new mineral novograblenovite, (NH4,K)MgCl3·6H2O, was found on basaltic lava from the 2012–2013 Tolbachik fissure eruption at the Plosky Tolbachik volcano, Kamchatka Peninsula, Russia. It occurs as prismatic, needle-like transparent crystals together with gypsum and halite. Novograblenovite was formed due to the exposure of the host rocks to eruptive gas exhalations enriched in HCl and NH3. Basalt was the source of potassium and magnesium for the mineral formation. Novograblenovite crystallises in the monoclinic space group C2/c, with unit-cell parameters a = 9.2734(3) Å, b = 9.5176(3) Å, c = 13.2439(4) Å, β = 90.187(2)°, V = 1168.91(2) Å3 and Z = 4. The five strongest reflections in the powder X-ray diffraction pattern [dobs, Å (I, %) (h k l)] are: 3.330 (100) (2 2 0), 2.976 (45) ($\bar{1}\; 1\; 4$), 2.353 (29) ($\bar {2}\; 2\; 4$), 3.825 (26) (2 0 2), 1.997 (25) ($\overline {4\; 2} $ 2). The density calculated from the empirical formula and the X-ray data is 1.504 g cm–3. The mineral is biaxial (+) with α = 1.469(2), β = 1.479(2) and γ = 1.496(2) (λ = 589 nm); 2Vmeas. = 80(10)° and 2Vcalc. = 75.7°. The crystal structure (solved and refined using single-crystal X-ray diffraction data, R1 = 0.0423) is based on the perovskite-like network of (NH4,K)Cl6-octahedra sharing chlorine vertices, and comprises [Mg(H2O)6]2+ groups in framework channels. The positions of all independent H atoms were obtained by difference-Fourier techniques and refined isotropically. All oxygen, nitrogen and chlorine atoms are involved in the system of hydrogen bonding, acting as donors or acceptors. The formula resulting from the structure refinement is [(NH4)0.7K0.3]MgCl3·6H2O. The mineral is named after Prokopiy Trifonovich Novograblenov, one of the researchers of Kamchatka Peninsula, a teacher, naturalist, geographer and geologist.

Crystallization and preliminary X-ray diffraction studies of a Bowman–Birk inhibitor from Vigna unguiculata seeds

1999 ◽  
Vol 55 (11) ◽  
pp. 1920-1922 ◽  
Author(s):  
K. N. Rao ◽  
S. S. Hegde ◽  
R. J. Lewis ◽  
C. G. Suresh
Keyword(s):  
Vigna Unguiculata ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Citrate Phosphate

A Bowman–Birk type trypsin/chymotrypsin inhibitor isolated from Vigna unguiculata seeds has been crystallized. Crystals were grown using the vapour-diffusion method at pH 4.0 using citrate/phosphate as a buffer and 30% saturated ammonium sulfate as precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 32.4, b = 61.8, c = 32.9 Å, β = 114.5°. The Matthews coefficient calculated assuming two molecules in the asymmetric unit was 1.95 Å3 Da−1, which corresponds to a 37% solvent content. X-ray data were collected to 2.5 Å resolution from a flash-frozen crystal. The structure was solved using the molecular-replacement method using tracy soybean inhibitor structure (PDB entry 1pi2) as a model.

scholarly journals Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) fromSinorhizobium meliloti

2014 ◽  
Vol 70 (2) ◽  
pp. 193-195 ◽  
Author(s):  
Bum Han Ryu ◽  
Duy Duc Nguyen ◽  
Tri Duc Ngo ◽  
Changsuk Oh ◽  
Ramesh Pandian ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Structure Refinement ◽  
Ammonium Phosphate ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Hydrolysis Of ◽  
Hydrolase Family

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) fromSinorhizobium melilotiwere performed. Recombinant Est24 protein containing an N-terminal His tag was expressed inEscherichia coliand purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 Mammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with anRmergeof 9.4%. The Est24 crystals belonged to space groupC2, with unit-cell parametersa= 129.09,b = 88.63,c= 86.15 Å, α = 90.00, β = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure ofMycobacterium smegmatisarylesterase as a template and structure refinement of Est24 is in progress.

scholarly journals Crystallization and preliminary X-ray analysis of a novel type of lipolytic hydrolase fromBacillus licheniformis

2014 ◽  
Vol 70 (4) ◽  
pp. 473-475
Author(s):  
Hansol Ju ◽  
Ramesh Pandian ◽  
Kyungmin Kim ◽  
Kyeong Kyu Kim ◽  
T. Doohun Kim
Keyword(s):  
Structure Refinement ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 4000 ◽  
Replacement Solution ◽  
Increasing Demand ◽  
Identification And Characterization

With increasing demand in biotechnological applications, the identification and characterization of novel lipolytic enzymes are of great importance. The crystallization and preliminary X-ray crystallographic study of a novel type of hydrolase fromBacillus licheniformis(BL28) are described here. Recombinant BL28 protein containing a C-terminal His tag was overproduced inEscherichia coliand purified to homogeneity. BL28 was crystallized using 0.2 Mammonium acetate, 0.1 Msodium citrate tribasic dihydrate pH 5.6, 30%(w/v) PEG 4000 as a crystallizing solution. X-ray diffraction data were collected to a resolution of 1.67 Å with anRmergeof 5.8%. The BL28 crystals belonged to the tetragonal space groupP41212, with unit-cell parametersa=b= 57.89,c= 167.25 Å. A molecular-replacement solution was obtained and structure refinement of BL28 is in progress.

scholarly journals Crystallographic studies of SarV, a global regulator fromStaphylococcus aureus

2015 ◽  
Vol 71 (8) ◽  
pp. 1038-1041
Author(s):  
Yang Song ◽  
Fan Zhang ◽  
Xu Li ◽  
Jianye Zang ◽  
Xuan Zhang
Keyword(s):  
Heavy Atom ◽  
Size Exclusion ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Global Regulator ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Heavy Atom Method

SarV, a member of the SarA protein family, is a global transcriptional regulator which has been reported to be involved in the regulation of autolysis inStaphylococcus aureus. In this study, SarV fromS. aureuswas successfully cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystals of SarV belonged to the monoclinic space groupP21, with unit-cell parametersa= 36.40,b= 119.64,c= 66.80 Å, α = γ = 90, β = 98.75°. The Matthews coefficient and the solvent content were estimated to be 2.57 Å3 Da−1and 52%, respectively, suggesting the presence of four molecules in the asymmetric unit. The results of size-exclusion chromatography (SEC) indicated thatS. aureusSarV exists as a homodimer in solution. Unfortunately, the structure cannot be solved by molecular replacement because of the low sequence identity ofS. aureusSarV to known structures. Further phase determination by selenomethionine single-wavelength anomalous dispersion (SAD) and the heavy-atom method is in progress.

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