scholarly journals Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

2015 ◽  
Vol 71 (9) ◽  
pp. 1156-1160 ◽  
Author(s):  
Tong Huo ◽  
Xiang Liu
Keyword(s):  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Replication Process ◽  
Reading Frame ◽  
X Ray ◽  
Cell Parameters ◽  
Human Coronavirus 229E ◽  
Important Enzyme

Nonstructural protein 15 (nsp15), also called endoribonuclease, is a gene product of open reading frame 1b (ORF 1b) in coronaviruses. It is an important enzyme in the transcription/replication process involved in discontinuous negative-strand RNA synthesis. In this work, mutants of nsp15 fromHuman coronavirus 229E(HCoV-229E) were made based on structural analysis of the homologous nsp15s inSevere acute respiratory syndrome coronavirus(SARS-CoV) andMouse hepatitis virus(MHV). The I26A/N52A mutant of nsp15 was overexpressed, purified and crystallized, and this mutant led to a trimeric form rather than hexamers or monomers. Crystals of trimeric nsp15 were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant and diffracted to 2.5 Å resolution. The crystals belonged to space groupC2221, with unit-cell parametersa= 85.9,b= 137.5,c= 423.1 Å, α = β = γ = 90°.

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 Cloning, purification and preliminary X-ray crystallographic analysis of the OmpA-like domain of peptidoglycan-associated lipoprotein fromAcinetobacter baumannii

2012 ◽  
Vol 68 (11) ◽  
pp. 1351-1353 ◽  
Author(s):  
Jung Hyun Song ◽  
Woo Cheol Lee ◽  
Jeong Soon Park ◽  
Seung Il Kim ◽  
Je Chul Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion ◽  
Strain Bl21

Peptidoglycan-associated lipoprotein (Pal) is one component of the Tol–Pal system that is involved in maintaining the integrity and stability of the outer membrane. The C-terminal OmpA-like domain of Pal interacts noncovalently with peptidoglycan. In this study, the OmpA-like domain of Pal fromAcinetobacter baumanniiwas overexpressed inEscherichia colistrain BL21 (DE3), purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.4 Å resolution and belonged to space groupP61orP65, with unit-cell parametersa=b= 72.58,c= 44.65 Å, a calculated Matthews coefficient of 2.64 Å3 Da−1and one molecule per asymmetric unit.

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 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 Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B fromGeobacillus collagenovoransMO-1

2012 ◽  
Vol 68 (7) ◽  
pp. 757-759 ◽  
Author(s):  
Hiroaki Nakano ◽  
Allin Hosokawa ◽  
Ryuji Tagawa ◽  
Koji Inaka ◽  
Kazunori Ohta ◽  
...  
Keyword(s):  
Space Station ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Peptide Substrates ◽  
X Ray ◽  
Cell Parameters ◽  
Experiment Module ◽  
Counter Diffusion

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophileGeobacillus collagenovoransMO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space groupP3121, with unit-cell parametersa=b= 87.64,c= 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

scholarly journals Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) fromOryza sativaL.japonica

2014 ◽  
Vol 70 (6) ◽  
pp. 781-785 ◽  
Author(s):  
Hackwon Do ◽  
Il-Sup Kim ◽  
Young-Saeng Kim ◽  
Sun-Young Shin ◽  
Jin-Ju Kim ◽  
...  
Keyword(s):  
Crop Production ◽  
Dehydroascorbate Reductase ◽  
Diffusion Method ◽  
Oxygen Species ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Key Enzyme ◽  
Important Enzyme

Dehydroascorbate reductase fromOryza sativaL.japonica(OsDHAR), a key enzyme in the regeneration of vitamin C, maintains reduced pools of ascorbic acid to detoxify reactive oxygen species. In previous studies, the overexpression of OsDHAR in transgenic rice increased grain yield and biomass as well as the amount of ascorbate, suggesting that ascorbate levels are directly associated with crop production in rice. Hence, it has been speculated that the increased level of antioxidants generated by OsDHAR protects rice from oxidative damage and increases the yield of rice grains. However, the crystal structure and detailed mechanisms of this important enzyme need to be further elucidated. In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 Mpotassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Å on beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space groupP21with unit-cell parametersa= 47.03,b= 48.38,c= 51.83 Å, β = 107.41°.

scholarly journals Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) fromDermatophagoides farinae

2015 ◽  
Vol 71 (11) ◽  
pp. 1396-1400 ◽  
Author(s):  
Sze Lei Pang ◽  
Kok Lian Ho ◽  
Jitka Waterman ◽  
Aik-Hong Teh ◽  
Fook Tim Chew ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
High Sequence Identity ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Dermatophagoides farinaeis one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in anEscherichia coliexpression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 MTris–HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to theC-centered monoclinic space groupC2, with unit-cell parametersa= 123.46,b= 27.71,c= 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å3 Da−1suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.

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.

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 Cloning, expression, purification, crystallization and X-ray crystallographic analysis ofD-lactate dehydrogenase fromLactobacillus jensenii

2014 ◽  
Vol 70 (8) ◽  
pp. 1046-1048 ◽  
Author(s):  
Sangwoo Kim ◽  
Yong Hwan Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Polyethylene Glycol 400 ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Lactobacillus Jensenii ◽  
Key Enzyme

The thermostable D-lactate dehydrogenase fromLactobacillus jensenii(LjD-LDH) is a key enzyme for the production of the D-form of lactic acid from pyruvate concomitant with the oxidation of NADH to NAD+. The polymers of lactic acid are used as biodegradable bioplastics. TheLjD-LDH protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 28%(w/v) polyethylene glycol 400, 100 mMTris–HCl pH 9, 200 mMmagnesium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.1 Å. The crystal belonged to space groupP3121, with unit-cell parametersa=b= 90.5,c= 157.8 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.58 Å3 Da−1, which corresponds to a solvent content of approximately 52.3%. The structure was solved by single-wavelength anomalous dispersion using a selenomethionine derivative.

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