scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of a soluble variant of the monoglyceride lipase Yju3p from the yeastSaccharomyces cerevisiae

2015 ◽  
Vol 71 (2) ◽  
pp. 243-246 ◽  
Author(s):  
Srinivasan Rengachari ◽  
Philipp Aschauer ◽  
Christian Sturm ◽  
Monika Oberer
Keyword(s):  
Crystal Form ◽  
Size Exclusion ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Monoglyceride Lipase ◽  
Exclusion Chromatography

The protein Yju3p is the orthologue of monoglyceride lipases in the yeastSaccharomyces cerevisiae. A soluble variant of this lipase termed s-Yju3p (38.3 kDa) was generated and purified to homogeneity by affinity and size-exclusion chromatography. s-Yju3p was crystallized in a vapour-diffusion setup at 293 K and a complete data set was collected to 2.4 Å resolution. The crystal form was orthorhombic (space groupP212121), with unit-cell parametersa= 77.2,b= 108.6,c= 167.7 Å. The asymmetric unit contained four molecules with a solvent content of 46.4%.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 fromRalstonia eutrophaH16

2015 ◽  
Vol 71 (6) ◽  
pp. 758-762
Author(s):  
Jieun Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Ralstonia Eutropha ◽  
Size Exclusion ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Fourth Step

The gene product of A1887 fromRalstonia eutropha(ReH16_A1887) has been annotated as a 3-ketoacyl-CoA thiolase, an enzyme that catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA.ReH16_A1887 was overexpressed and purified to homogeneity by affinity and size-exclusion chromatography. The degradative thiolase activity of the purifiedReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, withKm,Vmaxandkcatvalues of 158 µM, 32 mM min−1and 5 × 106 s−1, respectively. TheReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 MHEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space groupP43212, with unit-cell parametersa=b= 129.52,c= 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.

scholarly journals Protein purification, crystallization and preliminary X-ray diffraction analysis ofL-arabinose isomerase fromLactobacillus fermentumCGMCC2921

2015 ◽  
Vol 71 (1) ◽  
pp. 28-33 ◽  
Author(s):  
Zheng Xu ◽  
Sha Li ◽  
Jinfeng Liang ◽  
Xiaohai Feng ◽  
Hong Xu
Keyword(s):  
Monomethyl Ether ◽  
Size Exclusion ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Arabinose Isomerase

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose, as well as that of D-galactose to D-tagatose. A thermophilic AI derived fromLactobacillus fermentumCGMCC2921 (LFAI) was overexpressed inEscherichia coliBL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 Mbis-tris pH 6.5, 23% PEG 3350, 0.3 MNaCl (form 1 crystals) or 0.1 Mbis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 85.11,b= 184.57,c= 186.26 Å, α = β = γ = 90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da−1and a solvent content of 46.22%.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

2014 ◽  
Vol 70 (10) ◽  
pp. 1362-1367 ◽  
Author(s):  
Emmanuel Nji ◽  
Dianfan Li ◽  
Declan A. Doyle ◽  
Martin Caffrey
Keyword(s):  
Metal Ion ◽  
Cell Volume Regulation ◽  
Ion Concentration ◽  
Size Exclusion ◽  
Substrate Selectivity ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography

The prokaryotic lysine-specific permease (LysP) belongs to the amino acid–polyamine–organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP fromPseudomonas aeruginosawas recombinantly expressed inEscherichia coliand purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space groupP21, with unit-cell parametersa= 169.53,b= 169.53,c= 290.13 Å, γ = 120°.

scholarly journals Crystallization and preliminary X-ray diffraction studies of a novel bacterial esterase

1999 ◽  
Vol 55 (4) ◽  
pp. 915-917 ◽  
Author(s):  
Philip C. Bourne ◽  
Michail N. Isupov ◽  
Jennifer A. Littlechild
Keyword(s):  
Vapour Phase ◽  
Crystal Form ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Phase Calculation

A novel bacterial esterase has been crystallized in two forms suitable for X-ray diffraction studies. Crystals have been obtained by vapour-phase diffusion at 290 K using ammonium sulfate as precipitant. The first crystals grew in space group C2 with unit-cell parameters a = 134.7, b = 55.8, c = 110.3 Å, β = 125.1°. A monoclinic data set has been collected to 2.0 Å resolution. Microseeding yielded a second crystal form which grew in space group P212121 with unit-cell parameters a = 57.1, b = 115.4, c = 130.4 Å. Native data from these crystals have been collected to 1.6 Å resolution. A molecular envelope has been determined using an uranyl acetate derivative for phase calculation.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of UspE fromEscherichia coli

2014 ◽  
Vol 70 (12) ◽  
pp. 1640-1642 ◽  
Author(s):  
Yongbin Xu ◽  
Chun-Shan Quan ◽  
Xuanzhen Jin ◽  
Xiaoling Jin ◽  
Jing Zhao ◽  
...  
Keyword(s):  
Stress Proteins ◽  
Gel Filtration ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Vapour Diffusion ◽  
Induced Genes

Universal stress proteins (Usps) are among the most highly induced genes when bacteria are subjected to several stress conditions such as heat shock, nutrient starvation or the presence of oxidants or other stress agents.Escherichia colihas five small Usps and one tandem-type Usp. UspE (or YdaA) is the tandem-type Usp and consists of two Usp domains arranged in tandem. To date, the structure of UspE remains to be elucidated. To contribute to the molecular understanding of the function of the tandem-type UspE, UspE fromE. coliwas overexpressed and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. Crystals of UspE were obtained by sitting-drop vapour diffusion. A diffraction data set was collected to a resolution of 3.2 Å from flash-cooled crystals. The crystals belonged to the tetragonal space groupI4122 orI4322, with unit-cell parametersa=b= 121.1,c = 241.7 Å.

scholarly journals Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 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%.

Preparation and preliminary X-ray diffraction studies of a new crystal form of L-asparaginase from Escherichia coli

1999 ◽  
Vol 55 (9) ◽  
pp. 1616-1617
Author(s):  
I. Polikarpov ◽  
R. T. de Oliveira ◽  
J. Abrahão-Neto
Keyword(s):  
Escherichia Coli ◽  
Synchrotron Radiation ◽  
Crystal Form ◽  
Asymmetric Unit ◽  
Chemotherapeutic Drug ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Synchrotron Radiation Diffraction

L-Asparaginase is an enzyme which hydrolyzes asparagine to produce aspartic acid and ammonia. It is an effective chemotherapeutic drug, especially in the treatment of acute lymphoblastic leukaemia in children. The enzyme from Escherichia coli was crystallized in a new crystal form with space group C2, unit-cell parameters a = 76.3 (0), b = 134.6 (2), c = 64.8 (7) Å, β = 110.5 (1)° and a dimer in the asymmetric unit. Synchrotron-radiation diffraction data have been collected to 1.95 Å resolution.

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.

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.

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