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 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 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%.

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 High-resolution structure of an α-spectrin SH3-domain mutant with a redesigned hydrophobic core

2010 ◽  
Vol 66 (9) ◽  
pp. 1023-1027 ◽  
Author(s):  
Ana Cámara-Artigas ◽  
Monserrat Andújar-Sánchez ◽  
Emilia Ortiz-Salmerón ◽  
Celia Cuadri ◽  
Eva S. Cobos ◽  
...  
Keyword(s):  
Structural Information ◽  
Sh3 Domain ◽  
Hydrophobic Core ◽  
Orthorhombic Space Group ◽  
Cell Parameters ◽  
High Resolution Structure ◽  
Model Protein ◽  
Distal Loop ◽  
Modular Domain ◽  
The Stability

The α-spectrin SH3 domain (Spc-SH3) is a small modular domain which has been broadly used as a model protein in folding studies and these studies have sometimes been supported by structural information obtained from the coordinates of Spc-SH3 mutants. The structure of B5/D48G, a multiple mutant designed to improve the hydrophobic core and as a consequence the protein stability, has been solved at 1 Å resolution. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 24.79,b= 37.23,c= 62.95 Å. This mutant also bears a D48G substitution in the distal loop and this mutation has also been reported to increase the stability of the protein by itself. The structure of the B5/D48G mutant shows a highly packed hydrophobic core and a more ordered distal loop compared with previous Spc-SH3 structures.

Crystal structure of the GTP-binding protein-like domain of AGAP1

2021 ◽  
Vol 77 (4) ◽  
Author(s):  
Nuo Cheng ◽  
Hao Zhang ◽  
Shiyan Zhang ◽  
Xiaodan Ma ◽  
Guoyu Meng
Keyword(s):  
Membrane Trafficking ◽  
Protein Transport ◽  
Structural Information ◽  
Binding Protein ◽  
Lymphoblastic Leukemia ◽  
Diffusion Method ◽  
Gtp Binding Protein ◽  
Aberrant Expression ◽  
Cell Parameters ◽  
Gtp Binding

AGAP1 is often considered to regulate membrane trafficking, protein transport and actin cytoskeleton dynamics. Recent studies have shown that aberrant expression of AGAP1 is associated with many diseases, including neurodevelopmental disorders and acute lymphoblastic leukemia. It has been proposed that the GTP-binding protein-like domain (GLD) is involved in the binding of cofactors and thus regulates the catalytic activity of AGAP1. To obtain a better understanding of the pathogenic mechanism underpinning AGAP1-related diseases, it is essential to obtain structural information. Here, the GLD (residues 70–235) of AGAP1 was overexpressed in Escherichia coli BL21 (DE3) cells. Affinity and gel-filtration chromatography were used to obtain AGAP1GLD with high purity for crystallization. Using the hanging-drop vapor-diffusion method with the protein at a final concentration of 20 mg ml−1, AGAP1GLD protein crystals of suitable size were obtained. The crystals were found to diffract to 3.0 Å resolution and belonged to space group I4, with unit-cell parameters a = 100.39, b = 100.39, c = 48.08 Å. The structure of AGAP1GLD exhibits the highly conserved functional G1–G5 loops and is generally similar to other characterized ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), implying an analogous function to Arf GAPs. Additionally, this study indicates that AGAP1 could be classified as a type of NTPase, the activity of which might be regulated by protein partners or by its other domains. Taken together, these results provide insight into the regulatory mechanisms of AGAP1 in cell signaling.

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.

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 Purification, crystallization and preliminary X-ray crystallographic studies of Rv3705c fromMycobacterium tuberculosis

2014 ◽  
Vol 70 (8) ◽  
pp. 1090-1092
Author(s):  
Feifei Lu ◽  
Feng Gao ◽  
Honglin Li ◽  
Weimin Gong ◽  
Lin Zhou ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Unit Cell ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

The conserved protein Rv3705c fromMycobacterium tuberculosishas been cloned, expressed, purified and crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The Rv3705c crystals exhibited space groupP6122 orP6522, with unit-cell parametersa=b= 198.0,c= 364.1 Å, α = β = 90, γ = 120°, and diffracted to a resolution of 3.3 Å.

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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