scholarly journals Expression, crystallization and preliminary X-ray crystallographic analysis of DNA-directed RNA polymerase subunit L fromThermococcus onnurineusNA1

2014 ◽  
Vol 70 (5) ◽  
pp. 639-642
Author(s):  
Thien-Hoang Ho ◽  
Myoung-Ki Hong ◽  
Ho-Phuong-Thuy Ngo ◽  
Lin-Woo Kang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Multiprotein Complex ◽  
Hanging Drop ◽  
Gene Encoding ◽  
X Ray ◽  
Cell Parameters ◽  
And Function

RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, theTON-0309gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) fromThermococcus onnurineusNA1 was cloned and the protein was overexpressed inEscherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.10 Å resolution. The crystal belonged to the hexagonal space groupP6122, with unit-cell parametersa=b= 42.3,c= 211.2 Å. One molecule was present in the asymmetric unit, with a correspondingVMof 2.5 Å3 Da−1and a solvent content of 50.0%.

scholarly journals Overexpression, crystallization and preliminary X-ray crystallographic analysis of hypothetical protein SAV0479 fromStaphylococcus aureusMu50

2013 ◽  
Vol 69 (4) ◽  
pp. 405-407
Author(s):  
Chinar Pathak ◽  
Sun-Bok Jang ◽  
Hookang Im ◽  
Hye-Jin Yoon ◽  
Bong-Jin Lee
Keyword(s):  
Hypothetical Protein ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Vancomycin Resistant ◽  
And Function

SAV0479, a hypothetical protein from the Mu50 strain of methicillin- and vancomycin-resistantStaphylococcus aureus, was selected for structure and function determination as part of a structural genomics project. Here, the cloning, overexpression, purification and crystallization of SAV0479 are reported. Crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 Å. The crystals belonged to space groupP3121, with unit-cell parametersa=b= 81.48,c= 82.53 Å. Three monomers of SAV0479 are present in each asymmetric unit.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of the ArsM arsenic(III)S-adenosylmethionine methyltransferase

2010 ◽  
Vol 66 (9) ◽  
pp. 1050-1052 ◽  
Author(s):  
Kavitha Marapakala ◽  
A. Abdul Ajees ◽  
Jie Qin ◽  
Banumathi Sankaran ◽  
Barry P. Rosen
Keyword(s):  
Crystallographic Analysis ◽  
Hazardous Substances ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Priority List ◽  
X Ray ◽  
Cell Parameters ◽  
Environmental Toxin

Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III)S-adenosylmethionine methyltransferase, an enzyme (EC 2.1.1.137) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic algaCyanidioschyzonsp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 84.85,b= 46.89,c= 100.35 Å, β = 114.25° and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76 Å.

scholarly journals Crystallization and preliminary X-ray analysis of the hypothetical deaminase RPB_0146 fromRhodopseudomonas palustrisHaA2

2014 ◽  
Vol 70 (11) ◽  
pp. 1560-1562
Author(s):  
Guofang Zhang ◽  
Dan Yu ◽  
Guodong Yang ◽  
Hui Dong ◽  
Tongcun Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodopseudomonas Palustris ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

RPB_0146, a putative deaminase fromRhodopseudomonas palustrisHaA2, was expressed inEscherichia coliBL21 (DE3) cells and purified using a His6tag by Ni2+-chelating affinity chromatography for X-ray crystallographic analysis. Diffraction-quality crystals were grown by the hanging-drop vapour-diffusion method at 289 K and diffracted to a resolution of 2.44 Å using a wavelength of 1.000 Å at the Photon Factory (KEK), Japan. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 66.26,b= 123.94,c= 155.95 Å.

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 Crystallization and preliminary X-ray study of biosynthetic alanine racemase fromPseudomonas aeruginosaPAO1

2014 ◽  
Vol 70 (12) ◽  
pp. 1616-1619 ◽  
Author(s):  
Honggang Zhou ◽  
Zhenzhen Li ◽  
Guofang Zhang ◽  
Shujing Xu ◽  
Zhaona Tang ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Anion Exchange Chromatography ◽  
Crystallographic Analysis ◽  
Exchange Chromatography ◽  
Alanine Racemase ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Biosynthetic alanine racemase (AlrPA) fromPseudomonas aeruginosaPAO1 carrying a His6tag was expressed inEscherichia coliBL21 (DE3) cells and purified by Ni2+-chelating affinity and anion-exchange chromatography for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289 K in a solution consisting of 4%(v/v) Tacsimate pH 5.0, 14%(w/v) polyethylene glycol 3350 with a protein concentration of 8 mg ml−1. The crystal diffracted to 2.76 Å resolution and belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 74.12,b= 76.97,c= 154.80 Å, α = β = γ = 90°.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI fromEscherichia coli

2014 ◽  
Vol 70 (9) ◽  
pp. 1256-1259
Author(s):  
Mohan Zhao ◽  
Li Wang ◽  
Heng Zhang ◽  
Yuhui Dong ◽  
Yong Gong ◽  
...  
Keyword(s):  
16S Rrna ◽  
Crystallographic Analysis ◽  
Fine Tuning ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
And Function ◽  
Decoding Fidelity

RsmI and RsmH are AdoMet-dependent methyltransferases that are responsible for the 2′-O-methylation andN4-methylation of C1402 ofEscherichia coli16S rRNA, respectively. Modification of this site has been found to play a role in fine-tuning the shape and function of the P-site to increase the decoding fidelity. It is interesting to study the mechanism by which C1402 can be methylated by both RsmI and RsmH. The crystal structure of RsmH in complex with AdoMet and cytidine has recently been determined and provided some implications forN4-methylation of this site. Here, the purification and crystallization of RsmI as well as its preliminary crystallographic analysis are reported. Co-crystallization of RsmI with AdoMet was carried out by the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 2.60 Å resolution on beamline 1W2B at BSRF. The crystal contained three molecules per asymmetric unit and belonged to space groupC2, with unit-cell parametersa= 121.9,b= 152.5,c= 54.2 Å, β = 93.4°.

Purification, crystallization and preliminary X-ray crystallographic analysis of a phospholipase A2 from Daboia russelli pulchella

1999 ◽  
Vol 55 (4) ◽  
pp. 925-926 ◽  
Author(s):  
Vikas Chandra ◽  
Akanksha Nagpal ◽  
A. Srinivasan ◽  
T. P. Singh
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
The Novel ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Class 1

Phospholipases are esterolytic enzymes which hydrolyze glycerophospholipids. The pharmacological efficiency of phospholipase A2 (PLA2) enzymes is reflected by their specificity towards a tissue or organ. The Russell's viper has been classified into two classes. Class 1 contains Viper russelli russelli, Viper russelli siamensis and Viper russelli formosensis, whereas class 2 contains Daboia russelli pulchella. The sequence identity between the PLA2s from these two classes is 47%. The novel PLA2 from Daboia russelli pulchella has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitating agent. Crystals belong to the orthorhombic space group C2221 with unit-cell parameters a = 77.01, b = 92.29, c = 76.90 Å and two molecules in the asymmetric unit. These crystals diffract to about 2.49 Å resolution using a rotating-anode source.

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 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.

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.

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