scholarly journals Crystallization and preliminary X-ray diffraction analysis of domain-chimericL-(2S,3S)-butanediol dehydrogenase

2014 ◽  
Vol 70 (4) ◽  
pp. 461-463
Author(s):  
Tomohito Shimegi ◽  
Takuji Ooyama ◽  
Takashi Ohtsuki ◽  
Genji Kurisu ◽  
Masami Kusunoki ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
X Ray ◽  
Enzymatic Function ◽  
Vapour Diffusion ◽  
The Stability ◽  
Butanediol Dehydrogenase

A domain-chimeric L-2,3-butanediol dehydrogenase (chimera L-BDH), which was designed to possess both theS-configuration specificity of L-BDH and the stability ofmeso-BDH, was constructed by exchanging the respective domains of these two BDHs. However, chimera L-BDH possessed a lower enzymatic function than expected based on the two original enzymes. To elucidate the causes of the decreased stability and substrate specificity, crystallization of the protein was performed. Chimera L-BDH was purified to homogeneityviaammonium sulfate fractionation and three column-chromatography steps, and was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space groupC2221, diffracted synchrotron radiation to 1.58 Å resolution and were most likely to contain two molecules in the asymmetric unit.

scholarly journals Plant-specific DUF1110 protein fromOryza sativa: expression, purification and crystallization

2016 ◽  
Vol 72 (6) ◽  
pp. 480-484 ◽  
Author(s):  
Kenichi Harada ◽  
Eiki Yamashita ◽  
Kento Inoue ◽  
Koji Yamaguchi ◽  
Toshimichi Fujiwara ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Vapour Diffusion ◽  
Domain Of Unknown Function

The Os01T0156300 protein fromOryza sativahas been classified into the domain of unknown function (DUF) family DUF1110. DUF1110 family members exist in monocotyledons but not in dicotyledons, and share no sequence identity with proteins for which structures have been reported. In this study, the Os01T0156300 protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.84 Å resolution. The crystal belonged to space groupP21, with unit-cell parametersa= 89.9,b= 89.8,c= 107.1 Å, β = 106.6°. The asymmetric unit was estimated to contain 6–11 molecules.

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 Expression, purification, crystallization and preliminary X-ray analysis of tannase fromLactobacillus plantarum

2013 ◽  
Vol 69 (4) ◽  
pp. 456-459 ◽  
Author(s):  
Mingbo Wu ◽  
Xiaohong Peng ◽  
Hua Wen ◽  
Qin Wang ◽  
Qianming Chen ◽  
...  
Keyword(s):  
Synchrotron Radiation ◽  
Gallic Acid ◽  
Diffusion Method ◽  
Ester Bond ◽  
Asymmetric Unit ◽  
Data Set ◽  
X Ray ◽  
Vapour Diffusion ◽  
Serine Esterases ◽  
Hydrolysis Of

Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase fromLactobacillus plantarumwas cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space groupP1, with unit-cell paramtersa= 46.5,b= 62.8,c= 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.

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%.

Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human spliceosomal protein U5-15kD

1999 ◽  
Vol 55 (4) ◽  
pp. 888-890 ◽  
Author(s):  
Klaus Reuter ◽  
Ralf Ficner
Keyword(s):  
Escherichia Coli ◽  
Data Collection ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Gene Coding ◽  
Vapour Diffusion ◽  
Spliceosomal Protein

The gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding. The crystals belong to the orthorhombic space group P21212 with a = 62.3, b = 65.7, c = 37.1 Å. They diffract to at least 3.0 Å and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection.

Crystallization and preliminary X-ray diffraction studies of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus

1999 ◽  
Vol 55 (7) ◽  
pp. 1353-1355 ◽  
Author(s):  
C. Charron ◽  
F. Talfournier ◽  
M. N. Isupov ◽  
G. Branlant ◽  
J. A. Littlechild ◽  
...  
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Data Set ◽  
Hyperthermophilic Archaeon ◽  
X Ray ◽  
Cell Dimensions ◽  
Methanothermus Fervidus ◽  
Unit Cell Dimensions

The homotetrameric holo-D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus has been crystallized in the presence of NADP+ using the hanging-drop vapour-diffusion method. Crystals grew from a solution containing 2-methyl-2,4-pentanediol and magnesium acetate. A native data set has been collected to 2.1 Å using synchrotron radiation and cryocooling. Diffraction data have been processed in the orthorhombic system (space group P21212) with unit-cell dimensions a = 136.7, b = 153.3, c = 74.9 Å and one tetramer per asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

1999 ◽  
Vol 55 (6) ◽  
pp. 1231-1233 ◽  
Author(s):  
Ivana Smatanová ◽  
Yuji Nagata ◽  
L. Anders Svensson ◽  
Masamichi Takagi ◽  
Jaromír Marek
Keyword(s):  
Diffusion Method ◽  
Sphingomonas Paucimobilis ◽  
Asymmetric Unit ◽  
Haloalkane Dehalogenase ◽  
X Ray Diffraction ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18–20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris–HCl pH 8.9. The crystals diffract to at least 1.60 Å using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 Å. The asymmetric unit contains one molecule of the enzyme.

scholarly journals Crystallization and X-ray analysis ofD-threonine aldolase fromChlamydomonas reinhardtii

2017 ◽  
Vol 73 (2) ◽  
pp. 86-89
Author(s):  
Yuki Hirato ◽  
Masaru Goto ◽  
Mayumi Tokuhisa ◽  
Minoru Tanigawa ◽  
Katsushi Nishimura
Keyword(s):  
Diffusion Method ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Threonine Aldolase ◽  
Vapour Diffusion ◽  
Resolution Analysis

D-Threonine aldolase from the green algaChlamydomonas reinhardtii(CrDTA) catalyzes the interconversion of several β-hydroxy-D-amino acids (e.g.D-threonine) and glycine plus the corresponding aldehydes. Recombinant CrDTA was overexpressed inEscherichia coliand purified to homogeneity; it was subsequently crystallized using the hanging-drop vapour-diffusion method at 295 K. Data were collected and processed at 1.85 Å resolution. Analysis of the diffraction pattern showed that the crystal belonged to space groupP1, with unit-cell parametersa= 64.79,b= 74.10,c= 89.94 Å, α = 77.07, β = 69.34, γ = 71.93°. The asymmetric unit contained four molecules of CrDTA. The Matthews coefficient was calculated to be 2.12 Å3 Da−1and the solvent content was 41.9%.

Preliminary X-ray crystallographic studies of a newly defined human theta-class glutathione transferase

1998 ◽  
Vol 54 (1) ◽  
pp. 148-150 ◽  
Author(s):  
William J. McKinstry ◽  
Aaron J. Oakley ◽  
Jamie Rossjohn ◽  
Denis Verger ◽  
Kian-Leong Tan ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Critical Role ◽  
Environmental Pollutants ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
X Ray ◽  
Cell Dimensions ◽  
Vapour Diffusion ◽  
Glutathione S Transferases

Human theta-class glutathione S-transferases (GST's) appear to play a critical role in the metabolism of a variety of environmental pollutants but in some cases the products of the reaction are carcinogenic. Crystals of a human theta-class GST, namely hGSTT2-2, have been grown from polyethylene glycol by the hanging-drop vapour-diffusion method. The crystals belong to the trigonal space group P3121 with cell dimensions of a = b = 94.0 and c = 120.5 Å. They contain two monomers in the asymmetric unit and diffract to 3.0 Å resolution.

scholarly journals Cloning, expression, crystallization and preliminary X-ray diffraction studies of staphylococcal superantigen-like protein 1 (SSL1)

2014 ◽  
Vol 70 (5) ◽  
pp. 600-603 ◽  
Author(s):  
Debabrata Dutta ◽  
Anirudha Dutta ◽  
Atanu Bhattacharjee ◽  
Amit Basak ◽  
Amit Kumar Das
Keyword(s):  
Expression Vector ◽  
Diffusion Method ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Vapour Diffusion ◽  
Structural Homologues

Staphylococcus aureusproduces a family of exotoxins which are structural homologues of superantigens and thus are called staphylococcal superantigen-like proteins (SSLs). Amongst the 14 SSL genes,ssl1(SAOUHSC_00383) has been cloned in the pQE30 expression vector, overexpressed inEscherichia coliM15 (pREP4) cells and the protein purified to homogeneity. The protein was crystallized using 6% Tacsimate pH 6.0, 0.1 MMES pH 6.0, 25%(w/v) polyethylene glycol 3350, 100 mMNDSB 256 at 298 K by the sitting-drop vapour-diffusion method. The crystals belonged to space groupP21, with unit-cell parametersa= 77.9,b= 70.5,c= 126.5 Å, β = 106.2°. X-ray diffraction data were collected and processed to a maximum resolution of 2.5 Å. The crystal contains six molecules in the asymmetric unit.

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