scholarly journals Overexpression, purification, crystallization and preliminary X-ray crystallographic characterization of the receiver domain of the response regulator PhoP from Enterococcus faecalis ATCC 29212

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Yoon Chae Jeong ◽  
Ki Seog Lee
Keyword(s):  
Enterococcus Faecalis ◽  
Response Regulator ◽  
Critical Role ◽  
Transcriptional Response ◽  
Diffusion Method ◽  
Two Component System ◽  
Orthorhombic Space Group ◽  
Cell Parameters ◽  
Receiver Domain

Abstract Phosphate (Pho) regulon plays a critical role in bacterial phosphate homeostasis. It is regulated by two-component system (TCS) that comprises a sensor histidine kinase and transcriptional response regulator (RR). PhoP from Enterococcus faecalis (EfPhoP) belongs to the OmpR subfamily of RRs. It has not yet been structurally characterized because it is difficult to crystallize it to full-length form. In this study, a truncated form of EfPhoP containing the receiver domain (EfPhoP-RD) was constructed, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystal of EfPhoP-RD diffracted to 3.5 Å resolution and belonged to the orthorhombic space group C2221, with unit-cell parameters a = 118.74, b = 189.83, c = 189.88 Å. The asymmetric unit contains approximately 12 molecules, corresponding to a Matthews coefficient (Vm) of 2.50  Å3 Da−1 with a solvent content of 50.9%.

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the receiver domain of PhoB

1998 ◽  
Vol 54 (6) ◽  
pp. 1460-1463 ◽  
Author(s):  
Maria Solà ◽  
F.-Xavier Gomis-Rüth ◽  
Alicia Guasch ◽  
Luis Serrano ◽  
Miquel Coll
Keyword(s):  
Response Regulator ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Signal Transduction System ◽  
Hanging Drop ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
E Coli ◽  
Receiver Domain ◽  
Two Component Signal Transduction

PhoB is the response regulator of the E. coli two-component signal transduction system for phosphate regulation. It is a transcription factor that activates more than 30 genes of the pho regulon. Crystals of the receiver domain of PhoB were obtained by applying the hanging-drop vapour-diffusion method. X-ray diffraction data have been collected using synchrotron radiation to 1.88 Å resolution. The crystals belong to the orthorhombic space group P212121 with unit-cell constants a = 34.11, b = 60.42, c = 119.97 Å. The Matthews parameter suggests that PhoB crystallizes with two molecules per asymmetric unit, suggesting that activating dimerization occurs in the crystal.

scholarly journals A proposed carbon-utilization and virulence protein A, CuvA (Rv1422), from Mycobacterium tuberculosis H37Rv: crystallization, X-ray diffraction analysis and ligand binding

2020 ◽  
Vol 76 (7) ◽  
pp. 314-319
Author(s):  
Yoon Chae Jeong ◽  
Ki Seog Lee
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Environmental Changes ◽  
Structural Information ◽  
Critical Role ◽  
Nutrient Utilization ◽  
Diffusion Method ◽  
Uridine Diphosphate ◽  
Carbon Utilization ◽  
Orthorhombic Space Group ◽  
Cell Parameters

Mycobacterium tuberculosis possesses the ability to undergo physiological adaptations in order to persist during the prolonged course of infection despite the active immune response of the host and in order to overcome multiple environmental changes. Previous studies have proposed that M. tuberculosis CuvA (Rv1422; MtCuvA) might play a critical role in the adaptation of the bacterium to environmental changes, such as nutrient utilization and alteration of the growth rate. However, the detailed function of MtCuvA still remains unclear owing to a lack of structural information. To better understand its role in host adaptation, MtCuvA was purified to homogeneity and was crystallized for the first time using the hanging-drop vapor-diffusion method. The crystal of MtCuvA diffracted to a resolution of 2.1 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The calculated Matthews coefficient (V M) was 2.4 Å3 Da−1, with a solvent content of 48.02%, and thus four molecules appeared to be present in the asymmetric unit. Moreover, it is reported that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.

scholarly journals Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC fromStreptomyces antibioticus

2012 ◽  
Vol 68 (11) ◽  
pp. 1378-1386 ◽  
Author(s):  
Michael R. Jackson ◽  
Thomas L. Selby
Keyword(s):  
X Ray Diffraction ◽  
Hanging Drop ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Streptomyces Antibioticus ◽  
Cell Parameters ◽  
Heavy Atoms ◽  
Hanging Drop Method

A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) fromStreptomyces antibioticushas been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space groupP222, with unit-cell parametersa= 41.26,b= 51.86,c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of PBPD2 fromListeria monocytogenes

2014 ◽  
Vol 70 (4) ◽  
pp. 535-537 ◽  
Author(s):  
Hyung Jin Cha ◽  
Jae-Hee Jeong ◽  
Yeon-Gil Kim
Keyword(s):  
Biosynthetic Pathway ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Low Molecular Weight ◽  
Molecular Replacement ◽  
Orthorhombic Space Group ◽  
Penicillin Binding Proteins ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

scholarly journals Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of a human condensin SMC2 hinge domain with short coiled coils

2010 ◽  
Vol 66 (9) ◽  
pp. 1067-1070 ◽  
Author(s):  
Kazuki Kawahara ◽  
Shota Nakamura ◽  
Yasuhiro Katsu ◽  
Daisuke Motooka ◽  
Yuki Hosokawa ◽  
...  
Keyword(s):  
Structural Information ◽  
Critical Role ◽  
Crystallographic Analysis ◽  
Coiled Coils ◽  
Orthorhombic Space Group ◽  
Electron Microscopic ◽  
Space Group ◽  
Cell Parameters ◽  
Microscopic Studies ◽  
Hinge Domain

In higher eukaryotes, the condensin complex, which mainly consists of two structural maintenance of chromosomes (SMC) subunits, SMC2 (CAP-E) and SMC4 (CAP-C), plays a critical role in the formation of higher order chromosome structures during mitosis. Biochemical and electron-microscopic studies have revealed that the SMC2 and SMC4 subunits dimerize through the interaction of their hinge domains, forming a characteristic V-shaped heterodimer. However, the details of their function are still not fully understood owing to a lack of structural information at the atomic level. In this study, the human SMC2 hinge domain with short coiled coils was cloned, expressed, purified and crystallized in the orthorhombic space groupC222 in native and SeMet-derivatized forms. Because of the poor diffraction properties of these crystals, the mutant Leu68→SeMet was designed and crystallized in order to obtain the experimental phases. The SeMet-derivatized crystals of the mutant belonged to space groupP3212, with unit-cell parametersa=b= 128.8,c = 91.4 Å. The diffraction data obtained from a crystal that diffracted to 2.4 Å resolution were suitable for SAD phasing.

scholarly journals Crystal Structure of the Response Regulator 02 Receiver Domain, the Essential YycF Two-Component System of Streptococcus pneumoniae in both Complexed and Native States

2004 ◽  
Vol 186 (9) ◽  
pp. 2872-2879 ◽  
Author(s):  
Colin J. Bent ◽  
Neil W. Isaacs ◽  
Timothy J. Mitchell ◽  
Alan Riboldi-Tunnicliffe
Keyword(s):  
Streptococcus Pneumoniae ◽  
Active Site ◽  
Response Regulator ◽  
Thermotoga Maritima ◽  
Two Component System ◽  
Environmental Signals ◽  
Receiver Domain ◽  
Two Component ◽  
Protein Sequence Homology ◽  
Two Component Signal Transduction

ABSTRACT A variety of bacterial cellular responses to environmental signals are mediated by two-component signal transduction systems comprising a membrane-associated histidine protein kinase and a cytoplasmic response regulator (RR), which interpret specific stimuli and produce a measured physiological response. In RR activation, transient phosphorylation of a highly conserved aspartic acid residue drives the conformation changes needed for full activation of the protein. Sequence homology reveals that RR02 from Streptococcus pneumoniae belongs to the OmpR subfamily of RRs. The structures of the receiver domains from four members of this family, DrrB and DrrD from Thermotoga maritima, PhoB from Escherichia coli, and PhoP from Bacillus subtilis, have been elucidated. These domains are globally very similar in that they are composed of a doubly wound α5β5; however, they differ remarkably in the fine detail of the β4-α4 and α4 regions. The structures presented here reveal a further difference of the geometry in this region. RR02 is has been shown to be the essential RR in the gram-positive bacterium S. pneumoniae R. Lange, C. Wagner, A. de Saizieu, N. Flint, J. Molnos, M. Stieger, P. Caspers, M. Kamber, W. Keck, and K. E. Amrein, Gene 237:223-234, 1999; J. P. Throup, K. K. Koretke, A. P. Bryant, K. A. Ingraham, A. F. Chalker, Y. Ge, A. Marra, N. G. Wallis, J. R. Brown, D. J. Holmes, M. Rosenberg, and M. K. Burnham, Mol. Microbiol. 35:566-576, 2000). RR02 functions as part of a phosphotransfer system that ultimately controls the levels of competence within the bacteria. Here we report the native structure of the receiver domain of RR02 from serotype 4 S. pneumoniae (as well as acetate- and phosphate-bound forms) at different pH levels. Two native structures at 2.3 Å, phased by single-wavelength anomalous diffraction (xenon SAD), and 1.85 Å and a third structure at pH 5.9 revealed the presence of a phosphate ion outside the active site. The fourth structure revealed the presence of an acetate molecule in the active site.

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 Purification and crystallization of yeast Ent1 ENTH domain

2012 ◽  
Vol 68 (7) ◽  
pp. 820-823 ◽  
Author(s):  
Neta Tanner ◽  
Gali Prag
Keyword(s):  
Protein Product ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Enth Domain ◽  
Coomassie Blue ◽  
Sds Page ◽  
Crystallographic Symmetry

Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed inEscherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space groupP222, with unit-cell parametersa= 32.7,b= 35.5,c= 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS–PAGE analysis of dissolved crystals.

scholarly journals Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds

2017 ◽  
Vol 106 (2) ◽  
pp. 223-235 ◽  
Author(s):  
Morgan E. Milton ◽  
C. Leigh Allen ◽  
Erik A. Feldmann ◽  
Benjamin G. Bobay ◽  
David K. Jung ◽  
...  
Keyword(s):  
Response Regulator ◽  
Receiver Domain

scholarly journals Crystallization and preliminary crystallographic analysis of the first condensation domain of viomycin synthetase

2013 ◽  
Vol 69 (4) ◽  
pp. 412-415 ◽  
Author(s):  
Kristjan Bloudoff ◽  
T. Martin Schmeing
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Peptide Bond Formation ◽  
Orthorhombic Space Group ◽  
Nonribosomal Peptide ◽  
Cell Parameters ◽  
Peptide Synthetases ◽  
Condensation Domain

Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular synthetic logic whereby each successive amino-acid monomer is added to the peptide chain by successive multi-domain modules. The condensation domain catalyzes the central chemical event in the synthetic cycle, peptide-bond formation, and is present in every elongation module of the NRPS. Viomycin is an antituberculosis nonribosomal peptide that is synthesized by a series of four NRPS proteins and then modified by tailoring proteins. In order to study the mechanisms of peptide-bond formation in viomycin and in NRPSs in general, a structural study of the first condensation domain of the viomycin synthetase protein VioA (VioA-C1) was initiated. The gene for VioA-C1 was cloned from genomic DNA ofStreptomyces vinaceus, expressed as an octahistidine-tagged construct and purified by column chromatography. VioA-C1 was crystallized using the sitting-drop vapor-diffusion method. X-ray diffraction data were collected on a rotating-anode source to 2.9 Å resolution. The data could be indexed in the orthorhombic space groupP212121, with unit-cell parametersa= 46.165,b= 68.335,c= 146.423 Å. There is likely to be one monomer in the asymmetric unit, giving a solvent content of 49.2% and a Matthews coefficient (VM) of 2.42 Å3 Da−1. Structural determination is in progress.

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