scholarly journals The periplasmic sensing domain ofVibrio fischerichemoreceptor protein A (VfcA): cloning, purification and crystallographic analysis

2016 ◽  
Vol 72 (5) ◽  
pp. 382-385 ◽  
Author(s):  
Abu Iftiaf Md Salah Ud-Din ◽  
Anna Roujeinikova
Keyword(s):  
Vibrio Fischeri ◽  
Protein A ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Euprymna Scolopes ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

Flagella-mediated motility and chemotaxis towards nutrients are important characteristics ofVibrio fischerithat play a crucial role in the development of its symbiotic relationship with its Hawaiian squid hostEuprymna scolopes. TheV. fischerichemoreceptor A (VfcA) mediates chemotaxis toward amino acids. The periplasmic sensory domain of VfcA has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 as a precipitating agent. The crystals belonged to space groupP1, with unit-cell parametersa = 39.9,b= 57.0,c= 117.0 Å, α = 88.9, β = 80.5, γ = 89.7°. A complete X-ray diffraction data set has been collected to 1.8 Å resolution using cryocooling conditions and synchrotron radiation.

Crystallization and preliminary X-ray diffraction studies on the N-utilizing substance-B (NusB) from Mycobacterium tuberculosis

2000 ◽  
Vol 56 (1) ◽  
pp. 64-66 ◽  
Author(s):  
B. Gopal ◽  
R. A. Cox ◽  
M. J. Colston ◽  
G. G. Dodson ◽  
S. J. Smerdon ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ribosomal Rna ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Core Complex ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

N-utilizing substance B (NusB) is a protein which forms part of a complex assembly in transcriptional antitermination in Mycobacterium tuberculosis. It forms a heterodimer with the product of the NusE gene (identical to the ribosomal protein S10) and mediates the process of transcriptional antitermination by forming the core complex with the nut site of the ribosomal RNA along with other protein factors. NusB has been cloned and overexpressed in Escherichia coli and crystallized using the hanging-drop vapour-diffusion method. The space group is P212121, with unit-cell parameters a = 46.6, b = 64.2, c = 90.1 Å. A native data set complete to 1.6 Å resolution has been collected from a single crystal.

scholarly journals Crystallization and preliminary X-ray diffraction analysis ofBacillus subtilisYwfE, anL-amino-acid ligase

2012 ◽  
Vol 68 (2) ◽  
pp. 203-206 ◽  
Author(s):  
Takeo Tsuda ◽  
Tomomi Suzuki ◽  
Shuichi Kojima
Keyword(s):  
Amino Acid ◽  
Diffusion Method ◽  
Dependent Manner ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters

Bacillus subtilisYwfE, an L-amino-acid ligase, catalyzes the formation of an α-dipeptide from L-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl2and the dipeptide L-Ala-L-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space groupP6522, with unit-cell parametersa=b= 90.85,c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.

scholarly journals Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of catfish (Clarias magur) haemoglobin

2012 ◽  
Vol 68 (11) ◽  
pp. 1371-1373
Author(s):  
S. M. Jaimohan ◽  
M. D. Naresh ◽  
A. B. Mandal
Keyword(s):  
Structural Basis ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Monoclinic System ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Substantial Interest

Haemoglobin is an interesting physiologically significant protein composed of specific functional prosthetic haem and globin moieties. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of fish haemoglobins (Hbs). Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out onClarias magurHb. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 2000 and NaCl as precipitants. The crystals belonged to the primitive monoclinic systemP2, with unit-cell parametersa= 98.35,b= 56.63,c= 112.88 Å, β = 100.22°; a complete data set was collected to a resolution of 2.4 Å. The Matthews coefficient of 2.42 Å3 Da−1for the crystal indicated the presence of two α2β2tetramers in the asymmetric unit.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 fromRalstonia eutrophaH16

2014 ◽  
Vol 70 (7) ◽  
pp. 955-958 ◽  
Author(s):  
Jieun Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Ralstonia Eutropha ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Dispersion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Crystal Volume

The (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 fromRalstonia eutropha(RePaaH1) is an enzyme used in the biosynthesis ofn-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to (S)-3-hydroxybutyryl-CoA. TheRePaaH1 protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 1.4 Mammonium sulfate, 0.1 Msodium cacodylate pH 6.0, 0.2 Msodium chloride at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.6 Å on a synchrotron beamline. The crystal belonged to space groupP3221, with unit-cell parametersa=b= 135.4,c= 97.2 Å. With three molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.68 Å3 Da−1, which corresponds to a solvent content of approximately 54.1%. The structure was solved by the single-wavelength anomalous dispersion method and refinement of the structure is in progress.

scholarly journals A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 117-121 ◽  
Author(s):  
Junmei Ding ◽  
Hujie Zhu ◽  
Yujia Ye ◽  
Jie Li ◽  
Nanyu Han ◽  
...  
Keyword(s):  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallization Solution ◽  
Gdsl Family

The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallization solution. X-ray diffraction data were collected to a resolution of 2.30 Å with an R merge of 16.4% from a crystal belonging to space group P41212 or P43212, with unit-cell parameters a = b = 68.50, c = 79.57 Å.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of the Csu pili CsuC–CsuA/B chaperone–major subunit pre-assembly complex fromAcinetobacter baumannii

2015 ◽  
Vol 71 (6) ◽  
pp. 770-774 ◽  
Author(s):  
Natalia Pakharukova ◽  
Minna Tuittila ◽  
Sari Paavilainen ◽  
Anton Zavialov
Keyword(s):  
Diffusion Method ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Gram Negative ◽  
X Ray ◽  
Cell Parameters ◽  
Abiotic Surfaces ◽  
Vapour Diffusion ◽  
Fimbrial Adhesins

The attachment of many Gram-negative pathogens to biotic and abiotic surfaces is mediated by fimbrial adhesins, which are assembledviathe classical, alternative and archaic chaperone–usher (CU) pathways. The archaic CU fimbrial adhesins have the widest phylogenetic distribution, yet very little is known about their structure and mechanism of assembly. To elucidate the biogenesis of archaic CU systems, structural analysis of the Csu fimbriae, which are used byAcinetobacter baumanniito form stable biofilms and cause nosocomial infection, was focused on. The major fimbriae subunit CsuA/B complexed with the CsuC chaperone was purified from the periplasm ofEscherichia colicells co-expressing CsuA/B and CsuC, and the complex was crystallized in PEG 3350 solution using the hanging-drop vapour-diffusion method. Selenomethionine-labelled CsuC–CsuA/B complex was purified and crystallized under the same conditions. The crystals diffracted to 2.40 Å resolution and belonged to the hexagonal space groupP6422, with unit-cell parametersa=b= 94.71,c = 187.05 Å, α = β = 90, γ = 120°. Initial phases were derived from a single anomalous diffraction (SAD) experiment using the selenomethionine derivative.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 fromVerticillium dahliae

2012 ◽  
Vol 68 (7) ◽  
pp. 802-805 ◽  
Author(s):  
Lei Han ◽  
Zheng Liu ◽  
Xinqi Liu ◽  
Dewen Qiu
Keyword(s):  
Pathogenic Fungus ◽  
Sodium Formate ◽  
Diffusion Method ◽  
Effector Protein ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Dispersion Method ◽  
Data Set ◽  
X Ray ◽  
Anomalous Signal

The effector protein PevD1 from the pathogenic fungusVerticillium dahliaewas purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 Msodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 Mpotassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.

scholarly journals Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase fromBacillus subtilis

2010 ◽  
Vol 66 (9) ◽  
pp. 1053-1055 ◽  
Author(s):  
Qianda Lu ◽  
Jinming Ma ◽  
Hui Rong ◽  
Jun Fan ◽  
Ye Yuan ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Gene Encoding ◽  
Data Set ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Strain Bl21 ◽  
Aminolaevulinic Acid Dehydratase

5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA ofBacillus subtilisand the protein was overexpressed inEscherichia colistrain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH– tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 Å.

scholarly journals Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase fromStreptomycessp. NRRL 2288

2014 ◽  
Vol 70 (6) ◽  
pp. 734-737 ◽  
Author(s):  
Lihan Zhang ◽  
Jing Chen ◽  
Takahiro Mori ◽  
Yan Yan ◽  
Wen Liu ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Antimycin A ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Unsaturated Acyl ◽  
Reductive Carboxylation

AntE fromStreptomycessp. NRRL 2288 is a crotonyl-CoA carboxylase/reductase that catalyzes the reductive carboxylation of various α,β-unsaturated acyl-CoAs to provide the building block at the C7 position for antimycin A biosynthesis. Recombinant AntE expressed inEscherichia coliwas crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 76.4,b= 96.7,c= 129.6 Å, α = β = γ = 90.0°. A diffraction data set was collected at the KEK Photon Factory to 2.29 Å resolution.

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