scholarly journals Crystallization and X-ray structure determination of a thermoalkalophilic lipase fromGeobacillusSBS-4S

2013 ◽  
Vol 69 (4) ◽  
pp. 355-357
Author(s):  
Muhammad Tayyab ◽  
Naeem Rashid ◽  
Clement Angkawidjaja ◽  
Shigenori Kanaya ◽  
Muhammad Akhtar
Keyword(s):  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Wide Range ◽  
Molecular Replacement Method

A thermoalkalophilic lipase (LIPSBS) from the newly isolatedGeobacillusstrain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBSusing the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 55.13,b= 71.75,c= 126.26 Å. The structure was determined at 1.6 Å resolution by the molecular-replacement method using the lipase fromG. stearothermophilusL1 as a model.

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.

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 Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis

2015 ◽  
Vol 71 (4) ◽  
pp. 466-470 ◽  
Author(s):  
Ruyi Ding ◽  
Cui Xu ◽  
Xu Chen ◽  
Mengyun Bao ◽  
Xiaoting Qiu
Keyword(s):  
Diffraction Analysis ◽  
Biosynthetic Pathway ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Antithrombotic Activity ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosawith a C-terminal His6tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space groupP4322, with unit-cell parametersa=b= 101.581,c= 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of PBPD2 fromListeria monocytogenes

2014 ◽  
Vol 70 (4) ◽  
pp. 535-537 ◽  
Author(s):  
Hyung Jin Cha ◽  
Jae-Hee Jeong ◽  
Yeon-Gil Kim
Keyword(s):  
Biosynthetic Pathway ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Low Molecular Weight ◽  
Molecular Replacement ◽  
Orthorhombic Space Group ◽  
Penicillin Binding Proteins ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

scholarly journals Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase fromOryza sativaL.japonica

2014 ◽  
Vol 70 (9) ◽  
pp. 1244-1248 ◽  
Author(s):  
Hackwon Do ◽  
Il-Sup Kim ◽  
Young-Saeng Kim ◽  
Sun-Young Shin ◽  
Jin-Ju Kim ◽  
...  
Keyword(s):  
Crop Yields ◽  
Diffusion Method ◽  
Monodehydroascorbate Reductase ◽  
Molecular Replacement ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Key Enzyme ◽  
Glutathione Cycle

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate–glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived fromOryza sativaL.japonicawas expressed usingEscherichia colistrain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Å resolution and contained one molecule in the asymmetric unit (the Matthews coefficientVMis 1.98 Å3 Da−1, corresponding to a solvent content of 38.06%) in space groupP41212 with unit-cell parametersa=b= 81.89,c= 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase fromAcidovoraxsp. strain KKS102 (PDB entry 4h4q) as a model.

scholarly journals Periplasmic form of dipeptidyl aminopeptidase IV fromPseudoxanthomonas mexicanaWO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis

2017 ◽  
Vol 73 (11) ◽  
pp. 601-606 ◽  
Author(s):  
Saori Roppongi ◽  
Chika Tateoka ◽  
Mayu Fujimoto ◽  
Ippei Iizuka ◽  
Saori Morisawa ◽  
...  
Keyword(s):  
Crystal Form ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Dpp Iv ◽  
Molecular Replacement Method ◽  
Dipeptidyl Aminopeptidase

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) fromPseudoxanthomonas mexicanaWO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1′-P2′…]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced inEscherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Å resolution were collected from a triclinic crystal form belonging to space groupP1, with unit-cell parametersa= 88.66,b= 104.49,c = 112.84 Å, α = 67.42, β = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method usingStenotrophomonas maltophiliaDPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.

The application of the molecular replacement method to the de novo determination of protein structure

1991 ◽  
Vol 24 (4) ◽  
pp. 399-424 ◽  
Author(s):  
Michael C. Lawrence
Keyword(s):  
Protein Structure ◽  
De Novo ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Electron Density Map ◽  
Novel Protein

The determination of a novel protein structure by X-ray diffraction is seldom straightforward. Three hurdles present themselves (i) the protein must be purified in sufficient quantity to allow crystallization trials, (ii) crystals must be grown to adequate size and must diffract to a resolution that will allow atomic detail to be revealed, and (iii) phases must be determined for the diffracted X-ray beams in order that an initial electron-density map may be calculated.

Determination of the Crystal Structure and Redefinition of Tsugaruite, Pb28As15S50Cl, the First Lead-Arsenic Chloro-Sulfosalt

2020 ◽  
Author(s):  
Cristian Biagioni ◽  
Luca Bindi ◽  
Koichi Momma ◽  
Ritsuro Miyawaki ◽  
Yoshitaka Matsushita ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Atomic Ratio ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Specific Position ◽  
Actual Classification ◽  
Aomori Prefecture

Abstract Tsugaruite was originally defined as a lead-arsenic sulfosalt from the Yunosawa mine, Aomori Prefecture, Japan. Until recently its crystal structure remained unsolved and its actual classification in the sulfosalt realm was unknown. Here the refinement of the crystal structure of tsugaruite using single-crystal X-ray diffraction data is reported. The mineral is orthorhombic, space group P2nn, with unit-cell parameters a = 8.0774(10), b = 15.1772(16), c = 38.129(4) Å, V = 4674.3(9) Å3, in agreement with previous studies. The solution of the crystal structure of this mineral revealed Cl occupying a specific position. Chlorine was thus sought and found using the electron microprobe; the average of six spot analyses gave (in wt.%): Pb 68.04, As 12.83, S 18.29, Cl 0.63, total 99.80. The empirical formula, calculated on the basis of Pb + As = 43 atoms per formula unit, is Pb28.26As14.74S49.08Cl1.52. Tsugaruite is an N = 4 plesiotypic derivative of the homologous series of Pb-Sb chloro-sulfosalts having the general formula Pb(2+2N)(Sb,Pb)(2+2N)S(2+2N)(S,Cl)(4+2N)ClN. It has a Cl/(Cl + S) atomic ratio close to that of other known Pb-Sb chloro-sulfosalts (pillaite, pellouxite) and slightly higher than that of dadsonite.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of the homing endonuclease I-CvuI fromChlorella vulgarisin complex with its target DNA

2014 ◽  
Vol 70 (2) ◽  
pp. 256-259 ◽  
Author(s):  
Pilar Redondo ◽  
Nekane Merino ◽  
Maider Villate ◽  
Francisco J. Blanco ◽  
Guillermo Montoya ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Homing Endonuclease ◽  
Homing Endonucleases ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
Strand Breaks ◽  
X Ray ◽  
Cell Parameters ◽  
Wide Range

Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of DNA. The engineering of these enzymes provides novel instruments for genome modification in a wide range of fields, including gene targeting, by inducing specific double-strand breaks. I-CvuI is a homing endonuclease from the green algaChlorella vulgaris. This enzyme was purified after overexpression inEscherichia coli. Crystallization experiments of I-CvuI in complex with its DNA target in the presence of Mg2+yielded crystals suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 62.83,b= 83.56,c= 94.40 Å. The self-rotation function and the Matthews coefficient suggested the presence of one protein–DNA complex per asymmetric unit. The crystals diffracted to a resolution limit of 1.9 Å using synchrotron radiation.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of PhaA fromRalstonia eutropha

2014 ◽  
Vol 70 (11) ◽  
pp. 1566-1569
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Ralstonia Eutropha ◽  
Condensation Reaction ◽  
Monomethyl Ether ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

Polyhydroxybutyrate (PHB) is a biopolymer that is in the spotlight because of its broad applications in bioplastics, fine chemicals, implant biomaterials and biofuels. PhaA fromRalstonia eutropha(RePhaA) is the first enzyme in the PHB biosynthetic pathway and catalyzes the condensation reaction of two acetyl-CoA molecules to give acetoacetyl-CoA.RePhaA was crystallized using the hanging-drop vapour-diffusion method in the presence of 20% polyethylene glycol monomethyl ether 2K, 0.1 MTris–HCl pH 8.5 and 0.2 MtrimethylamineN-oxide dihydrate at 295 K. X-ray diffraction data were collected to a maximum resolution of 1.96 Å on a synchrotron beamline. The crystal belonged to space groupP21, with unit-cell parametersa= 68.38,b= 105.47,c= 106.91 Å, α = γ = 90, β = 106.18°. With four subunits per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.3 Å3 Da−1, which corresponds to a solvent content of approximately 46.2%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

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