Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH.

ABSTRACT: EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 A. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.1 A(3) Da(-1), corresponding to 41.5% solvent content.

SUBMITTER: Raghunathan K

PROVIDER: S-EPMC2705639 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Expression, purification, crystallization and preliminary X-ray studies of Vibrio cholerae pseudopilin EpsH.

Raghunathan Kannan K Vago Frank S FS Ball Terry T Yakubova Nafissa N Grindem David D Wedemeyer William J WJ Arvidson Dennis N DN

Acta crystallographica. Section F, Structural biology and crystallization communications 20090627 Pt 7

EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 A. There were two protein molecules in the asymmetric unit, which gave a Ma ...[more]

PMID: 19574644

Similar Datasets

Cloning, expression, purification, crystallization and preliminary X-ray analysis of a fructokinase from Vibrio cholerae O395.

Project description:Fructokinase (FK), one of the crucial enzymes for sugar metabolism in bacterial systems, catalyses the unidirectional phosphorylation reaction from fructose to fructose 6-phosphate, thereby allowing parallel entry of fructose into glycolysis beside glucose. The cscK gene from Vibrio cholerae O395 coding for the enzyme FK has been cloned, overexpressed in Escherichia coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Crystals of V. cholerae FK (Vc-FK) and its cocrystal with fructose, adenosine diphosphate (ADP) and Mg2+ were grown in the presence of polyethylene glycol 6000 and diffracted to 2.45 and 1.75 Å resolution, respectively. Analysis of the diffraction data showed that both crystal forms have symmetry consistent with space group P2(1)2(1)2, but with different unit-cell parameters. Assuming the presence of two molecules in the asymmetric unit, the Matthews coefficient for the apo Vc-FK crystals was estimated to be 2.4 Å3 Da(-1), which corresponds to a solvent content of 48%. The corresponding values for the ADP- and sugar-bound Vc-FK crystals were 2.1 Å3 Da(-1) and 40%, respectively, assuming the presence of one molecule in the asymmetric unit.

| S-EPMC3509990 | biostudies-literature

Purification, crystallization and preliminary X-ray crystallographic analysis of TssL from Vibrio cholerae.

Project description:The type VI secretion system (T6SS) is a macromolecular complex that is conserved in Gram-negative bacteria. The T6SS secretes effector proteins into recipient cells in a contact-dependent manner in order to accomplish cooperative and competitive interactions with the cells. Although the composition and mechanism of the T6SS have been intensively investigated across many Gram-negative bacteria, to date structural information on T6SS components from the important pathogen Vibrio cholerae has been rare. Here, the cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the cytoplasmic domain of TssL, an inner membrane protein of the T6SS, from V. cholerae are reported. Diffraction data were collected to 1.5 Å resolution using synchrotron radiation. The crystal belonged to the hexagonal space group P61, with unit-cell parameters a = 78.4, b = 78.4, c = 49.5 Å. The successful structural characterization of TssL from V. cholerae will contribute to understanding the role of the membrane-associated subunits of the T6SS in more detail.

| S-EPMC4157432 | biostudies-literature

Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395.

Project description:Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1-CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, β = 96.5°, whereas crystals grown in space group P3(2)21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°.

| S-EPMC3232161 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray structure analysis of Vibrio cholerae uridine phosphorylase in complex with thymidine.

Project description:A high-resolution structure of the complex of Vibrio cholerae uridine 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 of VchUPh 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 the VchUPh-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?µl VchUPh (15?mg?ml(-1)), 1?µl 0.1?M thymidine and 1.5?µl reservoir solution [15%(w/v) PEG 4000, 0.2?M MgCl(2).6H2O in 0.1?M Tris-HCl pH 8.5]. The crystals diffracted to 2.12?Å resolution and belonged to space group P2(1) (No. 4), with unit-cell parameters a=91.80, b=95.91, c=91.89?Å, ?=119.96°. The Matthews coefficient was calculated as 2.18?Å3?Da(-1); the corresponding solvent content was 43.74%.

| S-EPMC3515389 | biostudies-literature

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDa Vibrio cholerae heat-shock protein VcHsp31.

Project description:The Gram-negative bacterium Vibrio cholerae, which is responsible for the diarrhoeal disease cholera in humans, induces the expression of numerous heat-shock genes. VcHsp31 is a 31 kDa putative heat-shock protein that belongs to the DJ-1/PfpI superfamily, functioning as both a chaperone and a protease. VcHsp31 has been cloned, overexpressed and purified by Ni(2+)-NTA affinity chromatography followed by gel filtration. Crystals of VcHsp31 were grown in the presence of PEG 6000 and MPD; they belonged to space group P2(1) and diffracted to 1.9 Å resolution. Assuming the presence of six molecules in the asymmetric unit, the Matthews coefficient was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%.

| S-EPMC3212456 | biostudies-literature

Purification, crystallization and preliminary X-ray analysis of the periplasmic haem-binding protein HutB from Vibrio cholerae.

Project description:The mechanism of haem transport across the inner membrane of pathogenic bacteria is currently insufficiently understood at the molecular level and no information is available for this process in Vibrio cholerae. To obtain structural insights into the periplasmic haem-binding protein HutB from V. cholerae (VcHutB), which is involved in haem transport through the HutBCD haem-transport system, at the atomic level, VcHutB was cloned, overexpressed and crystallized using 1.6 M ammonium sulfate as a precipitant at pH 7.0. X-ray diffraction data were collected to 2.4 Å resolution on the RRCAT PX-BL-21 beamline at the Indus-2 synchrotron, Indore, India. The crystals belonged to space group P4₃2₁2, with unit-cell parameters a = b = 62.88, c = 135.8 Å. Matthews coefficient calculations indicated the presence of one monomer in the asymmetric unit, with an approximate solvent content of 45.02%. Molecular-replacement calculations with Phaser confirmed the presence of a monomer in the asymmetric unit.

| S-EPMC4388173 | biostudies-literature

Crystallization and preliminary X-ray study of TsiV3 from Vibrio cholerae.

Project description:The bacterial type VI secretion system (T6SS), a dynamic organelle, participates in microbial competition by transporting toxic effector molecules to neighbouring cells to kill competitors. TsiV3, a recently defined T6SS immunity protein in Vibrio cholerae, possesses self-protection against killing by T6SS predatory cells by directly binding to and inhibiting their effector protein VgrG-3. Structural information about TsiV3 could help to illuminate its specific mechanism. In this study, TsiV3 from V. cholerae was cloned, expressed and crystallized and single-crystal X-ray diffraction data sets were collected to a resolution of 2.55 Å. Specifically, the crystal belonged to space group P212121, with unit-cell parameters a = 73.3, b = 78.12, c = 106.18 Å. Matthews coefficient calculations indicated that the crystal may contain six TsiV3 molecules in one asymmetric unit, with a VM value of 2.25 Å(3) Da(-1) and a solvent content of 45.42%.

| S-EPMC3944696 | biostudies-literature

Purification, crystallization and preliminary X-ray analysis of the AAA+ ?54 activator domain of FlrC from Vibrio cholerae.

Project description:A ?(54)-dependent transcriptional activator FlrC containing an N-terminal regulatory domain, a central AAA(+) domain and a C-terminal DNA-binding domain has been implicated both in flagellar synthesis and enhanced intestinal colonization. FlrC is phosphorylated by the kinase FlrB at the regulatory domain and both nonphosphorylated and phosphorylated states of FlrC seem to be important for its functions. Oligomerization plays a key role in the functions of such transcriptional activators and the AAA(+) ?(54) interaction domain is critical in deciding the oligomerization state. Therefore, to obtain structural insights into FlrC at the atomic level, the AAA(+) ?(54) interaction domain of FlrC was cloned, overexpressed and crystallized using PEG 6000 as precipitant at pH 6.0, and diffraction data were collected to 2.8 Å resolution. Molecular-replacement calculations and subsequent refinement confirmed the presence of a closed heptamer in the asymmetric unit.

| S-EPMC3702329 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray studies of Lactobacillus jensenii enolase.

Project description:Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 A resolution. The crystals belonged to space group I4, with unit-cell parameters a=b=145.31, c=99.79 A. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8 A3 Da(-1), corresponding to 55.2% solvent content.

| S-EPMC2917297 | biostudies-literature

Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae.

Project description:Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni(2+)-NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P3(1) and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å(3) Da(-1) and 55.62%, respectively.

| S-EPMC3497980 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data