scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of ArsH fromSynechocystissp. strain PCC 6803

2014 ◽  
Vol 70 (4) ◽  
pp. 497-500 ◽  
Author(s):  
Xiao Zhang ◽  
Xi-Mei Xue ◽  
Yu Yan ◽  
Jun Ye
Keyword(s):  
Sinorhizobium Meliloti ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Flavin Mononucleotide ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Pcc 6803

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of anarsoperon. The function of thearsHgene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH fromSynechocystissp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space groupI4122, with unit-cell parametersa=b= 127.94,c= 65.86 Å and one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH fromSinorhizobium melilotiwill provide information about the oligomerization of ArsH.

scholarly journals Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) fromSynechococcussp. PCC 7942

2014 ◽  
Vol 70 (2) ◽  
pp. 203-205
Author(s):  
Asaithambi Killivalavan ◽  
Ningning Zhuang ◽  
Young Shik Park ◽  
Kon Ho Lee
Keyword(s):  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
X Ray Diffraction ◽  
Dispersion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Multi Wavelength ◽  
Pcc 7942

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacteriumSynechococcussp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 Mbis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 171.35,b= 77.99,c = 53.77 Å, β = 90.27°.

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.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (10) ◽  
pp. 1362-1367 ◽  
Author(s):  
Emmanuel Nji ◽  
Dianfan Li ◽  
Declan A. Doyle ◽  
Martin Caffrey
Keyword(s):  
Metal Ion ◽  
Cell Volume Regulation ◽  
Ion Concentration ◽  
Size Exclusion ◽  
Substrate Selectivity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

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.

Characterization of the sorbitol dehydrogenase SmoS from Sinorhizobium meliloti 1021

2021 ◽  
Vol 77 (3) ◽  
pp. 380-390
Author(s):  
MacLean G. Kohlmeier ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Ivan J. Oresnik
Keyword(s):  
Sinorhizobium Meliloti ◽  
Model Organism ◽  
Time Frame ◽  
Industrial Applications ◽  
Size Exclusion ◽  
Ligand Docking ◽  
X Ray Diffraction ◽  
X Ray ◽  
Exclusion Chromatography

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD+ as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Å resolution and those of a complex with sorbitol diffracted to 2.0 Å resolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

scholarly journals PsEst3, a new psychrophilic esterase from the Arctic bacterium Paenibacillus sp. R4: crystallization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (6) ◽  
pp. 367-372
Author(s):  
Hyun Kim ◽  
Ae Kyung Park ◽  
Jun Hyuck Lee ◽  
Seung Chul Shin ◽  
Hyun Park ◽  
...  
Keyword(s):  
Expression System ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
The Arctic ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Paenibacillus Sp ◽  
Exclusion Chromatography ◽  
And Function

Esterases are very useful biocatalysts in industry: they hydrolyze esters and split them into a carboxylic acid and an alcohol. The psychrophilic esterase PsEst3 was obtained from Paenibacillus sp. R4, which was isolated from the active layer of the permafrost in Council, Alaska. PsEst3 was successfully overexpressed using a psychrophilic chaperonin co-expression system and was purified by nickel-affinity and size-exclusion chromatography. Recombinant PsEst3 was crystallized at 290 K using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution. The crystal was determined to belong to space group P4132 or P4332, with unit-cell parameters a = b = c = 145.33 Å. Further crystallographic analysis needs to be conducted to investigate the structure and function of this esterase.

scholarly journals Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

2014 ◽  
Vol 70 (11) ◽  
pp. 1526-1528 ◽  
Author(s):  
G. Jagadeesan ◽  
P. Malathy ◽  
K. Gunasekaran ◽  
S. Harikrishna Etti ◽  
S. Aravindhan
Keyword(s):  
Structural Basis ◽  
Diffusion Method ◽  
Great Cormorant ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Phalacrocorax Carbo ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Substantial Interest

Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal systemP3121, with unit-cell parametersa=b= 55.64,c= 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

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 Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeastSaccharomyces cerevisiae

2015 ◽  
Vol 71 (2) ◽  
pp. 243-246 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Philipp Aschauer ◽  
Christian Sturm ◽  
Monika Oberer
Keyword(s):  
Crystal Form ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Monoglyceride Lipase ◽  
Exclusion Chromatography

The protein Yju3p is the orthologue of monoglyceride lipases in the yeastSaccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space groupP212121), with unit-cell parametersa= 77.2,b= 108.6,c= 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

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.

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