scholarly journals Expression, purification, crystallization and preliminary X-ray structure analysis ofVibrio choleraeuridine phosphorylase in complex with thymidine

2012 ◽  
Vol 68 (11) ◽  
pp. 1394-1397 ◽  
Author(s):  
Alexander A. Lashkov ◽  
Azat G. Gabdulkhakov ◽  
Igor I. Prokofev ◽  
Tatyana A. Seregina ◽  
Sergey E. Sotnichenko ◽  
...  
Keyword(s):  
Rational Design ◽  
Diffusion Method ◽  
Expression And Purification ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 4000 ◽  
High Resolution Structure ◽  
Reservoir Solution ◽  
Crystallization Solution ◽  
Resolution Structure

A high-resolution structure of the complex ofVibrio choleraeuridine phosphorylase (VchUPh) with its physiological ligand thymidine is important in order to determine the mechanism of the substrate specificity of the enzyme and for the rational design of pharmacological modulators. Here, the expression and purification ofVchUPh and the crystallization of its complex with thymidine are reported. Conditions for crystallization were determined with an automated Cartesian Dispensing System using The Classics, MbClass and MbClass II Suites crystallization kits. Crystals of theVchUPh–thymidine complex (of dimensions ∼200–350 µm) were grown by the sitting-drop vapour-diffusion method in ∼7 d at 291 K. The crystallization solution consisted of 1.5 µlVchUPh (15 mg ml−1), 1 µl 0.1 Mthymidine and 1.5 µl reservoir solution [15%(w/v) PEG 4000, 0.2 MMgCl2.6H2O in 0.1 MTris–HCl pH 8.5]. The crystals diffracted to 2.12 Å resolution and belonged to space groupP21(No. 4), with unit-cell parametersa = 91.80,b= 95.91,c= 91.89 Å, β = 119.96°. The Matthews coefficient was calculated as 2.18 Å3 Da−1; the corresponding solvent content was 43.74%.

Production, crystallization and preliminary X-ray analysis of the human integrin \alpha_1 I domain

1999 ◽  
Vol 55 (7) ◽  
pp. 1365-1367 ◽  
Author(s):  
Tiina A. Salminen ◽  
Yvonne Nymalm ◽  
Jussi Kankare ◽  
Jarmo Käpylä ◽  
Jyrki Heino ◽  
...  
Keyword(s):  
Large Scale ◽  
Cell Types ◽  
Diffusion Method ◽  
Scale Production ◽  
Data Set ◽  
Cell Parameters ◽  
Collagen Receptors ◽  
Large Scale Production ◽  
Peg 4000 ◽  
Reservoir Solution

Integrin α1β1 is one of the main collagen receptors in many cell types. A fast large-scale production, purification and crystallization method for the integrin α1 I domain is reported here. The α1 I domain was crystallized using the vapour-diffusion method with a reservoir solution containing a mixture of PEG 4000, sodium acetate, glycerol and Tris–HCl buffer. The crystals beong to the C2 space group, with unit-cell parameters a = 74.5, b = 81.9, c = 37.3 Å, α = γ = 90.0, β = 90.8°. The crystals diffract to 2.0 Å and a 94.2% complete data set to 2.2 Å has been collected from a single crystal with an R merge of 5.8%.

scholarly journals Pteridine glycosyltransferase from Chlorobium tepidum: crystallization and X-ray analysis

2017 ◽  
Vol 73 (11) ◽  
pp. 629-634
Author(s):  
Asaithambi Killivalavan ◽  
Young Shik Park ◽  
Kon Ho Lee
Keyword(s):  
Diffusion Method ◽  
Chlorobium Tepidum ◽  
Monoclinic Space Group ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Chromatographic Techniques ◽  
Multiple Wavelength ◽  
Reservoir Solution

The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2′-yl)-β-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Å resolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c = 105.92 Å, β = 120.5°.

scholarly journals Expression, high-pressure refolding, purification, crystallization and preliminary X-ray analysis of a novel single-strand-specific 3′–5′ exonuclease PhoExo I fromPyrococcus horikoshiiOT3

2014 ◽  
Vol 70 (8) ◽  
pp. 1076-1079 ◽  
Author(s):  
Ken-ichi Miyazono ◽  
Kanae Tsutsumi ◽  
Yoshizumi Ishino ◽  
Masaru Tanokura
Keyword(s):  
High Pressure ◽  
Inclusion Bodies ◽  
Single Strand ◽  
Diffusion Method ◽  
X Rays ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Reservoir Solution

PhoExo I is a single-strand-specific 3′–5′ exonuclease fromPyrococcus horikoshiiOT3 and is thought to be involved in a Thermococcales-specific DNA-repair pathway. The recombinant PhoExo I protein was produced as inclusion bodies inEscherichia colicells. Solubilization of the inclusion bodies was performed by the high-pressure refolding method and highly purified protein was subjected to crystallization by the sitting-drop vapour-diffusion method at 20°C. A crystal of PhoExo I was obtained in a reservoir solution consisting of 0.1 MTris–HCl pH 8.9, 27% PEG 6000 and diffracted X-rays to 1.52 Å resolution. The crystal of PhoExo I belonged to space groupH32, with unit-cell parametersa=b= 112.07,c= 202.28 Å. The crystal contained two PhoExo I molecules in the asymmetric unit.

scholarly journals Purification, crystallization and preliminary crystallographic investigation of FrnE, a disulfide oxidoreductase fromDeinococcus radiodurans

2014 ◽  
Vol 70 (11) ◽  
pp. 1540-1542 ◽  
Author(s):  
Lata Panicker ◽  
Hari Sharan Misra ◽  
Subhash Chandra Bihani
Keyword(s):  
Oxidative Stress ◽  
Disulfide Bonds ◽  
Deinococcus Radiodurans ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Disulfide Oxidoreductase ◽  
Reservoir Solution ◽  
Peg 8000

In prokaryotes, Dsb proteins catalyze the formation of native disulfide bonds through an oxidative folding pathway and are part of the cell machinery that protects proteins from oxidative stress.Deinococcus radioduransis an extremophile which shows unparalleled resistance to ionizing radiation and oxidative stress. It has a strong mechanism to protect its proteome from oxidative damage. The genome ofDeinococcusshows the presence of FrnE, a Dsb protein homologue that potentially provides the bacterium with oxidative stress tolerance. Here, crystallization and preliminary X-ray crystallographic analysis of FrnE fromD. radioduransare reported. Diffraction-quality single crystals were obtained using the hanging-drop vapour-diffusion method with reservoir solution consisting of 100 mMsodium acetate pH 5.0, 10% PEG 8000, 15–20% glycerol. Diffraction data were collected on an Agilent SuperNova system using a microfocus sealed-tube X-ray source. The crystal diffracted to 1.8 Å resolution at 100 K. The space group of the crystal was found to beP21221, with unit-cell parametersa= 47.91,b= 62.94,c= 86.75 Å, α = β = γ = 90°. Based on Matthews coefficient analysis, one monomer per asymmetric unit is present in the crystal, with a solvent content of approximately 45%.

scholarly journals Xylanase B fromClostridium cellulovorans743B: overexpression, purification, crystallization and X-ray diffraction analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 113-116 ◽  
Author(s):  
Daichi Nakajima ◽  
Akihiko Nagano ◽  
Toshiyuki Shibata ◽  
Reiji Tanaka ◽  
Kouichi Kuroda ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Catalytic Domain ◽  
Cellulosic Biomass ◽  
Diffusion Method ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 4000

Clostridium cellulovoransproduces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 Msodium acetate trihydrate, 0.1 MTris–HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Å resolution and belonged to space groupP212121, with unit-cell parametersa = 74.28,b= 77.55,c= 88.20 Å, α = β = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase fromC. cellulovorans.

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 Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase fromBacillus licheniformisATCC 14580

2014 ◽  
Vol 70 (11) ◽  
pp. 1513-1516 ◽  
Author(s):  
Mayte Conejero-Muriel ◽  
Ana Isabel Martínez-Gómez ◽  
Sergio Martínez-Rodríguez ◽  
Jose A. Gavira
Keyword(s):  
Potassium Thiocyanate ◽  
Amide Bond ◽  
Diffusion Method ◽  
Hydrolytic Cleavage ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Crystallization Solution ◽  
Amidohydrolase Superfamily

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1—C2amide bond of the five-membered hydantoin ring. Allantoinase fromBacillus licheniformis(AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work,B. licheniformisATCC 14580 allantoinase (AllBali) containing a C-terminal His6tag was overproduced inEscherichia coliand purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 Mpotassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with anRmergeof 29.2% from a crystal belonging to space groupP1211, with unit-cell parametersa= 54.93,b= 164.74,c= 106.89 Å, β = 98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM= 2.34 Å3 Da−1).

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 Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI fromBacillussp. HJB17

2017 ◽  
Vol 73 (11) ◽  
pp. 607-611
Author(s):  
Fang Lu ◽  
Bei Zhang ◽  
Yong Liu ◽  
Ying Song ◽  
Gangxing Guo ◽  
...  
Keyword(s):  
Unit Cell ◽  
Diffusion Method ◽  
Data Sets ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters

Phytases are phosphatases that hydrolyze phytates to less phosphorylatedmyo-inositol derivatives and inorganic phosphate. β-Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β-propeller phytase fromBacillussp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vapour-diffusion method in a condition consisting of 0.2 Msodium chloride, 0.1 MTris pH 8.5, 25%(w/v) PEG 3350 at 289 K. X-ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH-DI crystals belonged to space groupC121, with unit-cell parametersa = 156.84,b = 45.54,c = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH-DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1and a solvent content of 43.26%. Crystals of selenomethionine-substituted PhyH-DI belonged to space groupC2221, with unit-cell parametersa = 94.71,b= 97.03,c= 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1and a solvent content of 49.64%. Initial phases for PhyH-DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH-DI.

scholarly journals Crystallographic studies of aspartate racemase fromLactobacillus sakeiNBRC 15893

2015 ◽  
Vol 71 (8) ◽  
pp. 1012-1016 ◽  
Author(s):  
Tomomi Fujii ◽  
Takae Yamauchi ◽  
Makoto Ishiyama ◽  
Yoshitaka Gogami ◽  
Tadao Oikawa ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Low Temperatures ◽  
Diffusion Method ◽  
Lactobacillus Sakei ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Aspartate Racemase ◽  
Vapour Diffusion ◽  
Brewing Process ◽  
Resolution Structure

Aspartate racemase catalyzes the interconversion between L-aspartate and D-aspartate and belongs to the PLP-independent racemases. The enzyme from the lactic acid bacteriumLactobacillus sakeiNBRC 15893, isolated fromkimoto, is considered to be involved in D-aspartate synthesis during the brewing process of Japanese sake at low temperatures. The enzyme was crystallized at 293 K by the sitting-drop vapour-diffusion method using 25%(v/v) PEG MME 550, 5%(v/v) 2-propanol. The crystal belonged to space groupP3121, with unit-cell parametersa=b= 104.68,c= 97.29 Å, and diffracted to 2.6 Å resolution. Structure determination is under way.

Sign in / Sign up

Export Citation Format

Share Document