Preliminary X-ray Diffraction Analysis Research Articles

Preliminary X-ray diffraction and ligand-binding analyses of the N-terminal domain of hypothetical protein Rv1421 from Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active disease through reactivation. M. tuberculosis Rv1421 (MtRv1421) is a hypothetical protein that has been proposed to be involved innucleotide binding-related metabolism in cell-growth and cell-division processes. However, due to a lack of structural information, the detailed function of MtRv1421 remains unclear. In this study, a truncated N-terminal domain (NTD) of MtRv1421, which contains a Walker A/B-like motif, was purified and crystallized using PEG 400 as a precipitant. The crystal of MtRv1421-NTD diffracted to a resolution of 1.7 Å and was considered to belong to either the C-centered monoclinic space group C2 or the I-centered orthorhombic space group I222, with unit-cell parameters a = 124.01, b = 58.55, c = 84.87 Å, β=133.12° or a = 58.53, b = 84.86, c = 90.52 Å, respectively. The asymmetric units of the C2 or I222 crystals contained two or one monomers, respectively. Interms of the binding ability of MtRv1421-NTD to various ligands, uridine diphosphate (UDP) and UDP-N-acetylglucosamine significantly increased the melting temperature of MtRv1421-NTD, which indicates structural stabilization through the binding of these ligands. Altogether, the results reveal that a UDP moiety may be required for the interaction of MtRv1421-NTD as a nucleotide-binding protein with its ligand.

Preliminary X-ray Diffraction Analysis of Purine Nucleoside Phosphorylase from the Haloalkaliphilic Bacterium Halomonas chromatireducens

Preliminary X-ray Diffraction Analysis of Purine Nucleoside Phosphorylase from the Haloalkaliphilic Bacterium Halomonas chromatireducens

Preliminary X-ray Diffraction Analysis of Purine Nucleoside Phosphorylase from the Haloalkaliphilic Bacterium Halomonas chromatireducens

Crystals of the enzyme purine nucleoside phosphorylase from the extremophilic bacterium Halomonas Chromatireducens AGD 8-3, suitable for X-ray diffraction, were grown by the vapor-diffusion method. The X-ray diffraction data were collected from these crystals at the Belok beamline of the Kurchatov synchrotron radiation source (National Research Centre “Kurchatov Institute”) at 100 K to 1.80 Å resolution. The X-ray diffraction data were processed in the space groups P1, P2, P21, and P622. The structure was solved by the molecular replacement method taking into account the twinning in the space groups P21 and P1 with one and two hexamers of the enzyme per asymmetric unit, respectively.

Lead-free complex double perovskite SrLiFeWO6: Structural, microstructure, electrical and optical study

In this work, synthesis (solid-state reaction) and characterizations (structural, microstructure, dielectric, conductivity, Raman & UV–vis) of a newly prepared lead-free complex perovskite SrLiFeWO6 are reported. Preliminary X-ray diffraction (XRD) analysis suggests a tetragonal phase. The scanning electron microscope (SEM) micrograph and energy dispersive x-ray analysis (EDX) spectrum show the well-grown grains and sample purity. The study of Raman active lines determines the vibrational modes of the constituent molecules in the reported compound. Dispersed dielectric plots were explained by the Maxwell-Wagner polarization effect. The modulus study reveals the presence of a non-Debye type of relaxation. Jonscher's Power Law is fitted in ac conductivity data which follows a correlated barrier hopping mechanism. The direct bandgap energy of the studied sample is found to be 1.68 eV, which may be a good range for photovoltaic applications. For NTC thermistor applications, the analyzed thermistor constant and sensitivity factor are found to be within the permissible range. The idea of ferroelectric nature is opened up by the study of polarization versus electric field. The examination of current density versus electric field reveals that the sample has an ohmic character in the low region and a space charge-limited conduction (SCLC) mechanism in the higher field region.

Identification, Characterization, and Preliminary X-ray Diffraction Analysis of a Novel Esterase (ScEst) from Staphylococcus chromogenes

Ester prodrugs can develop novel antibiotics and have potential therapeutic applications against multiple drug-resistant bacteria. The antimicrobial activity of these prodrugs is activated after being cleaved by the esterases produced by the pathogen. Here, novel esterase ScEst originating from Staphylococcus chromogenes NCTC10530, which causes dairy cow mastitis, was identified, characterized, and analyzed using X-ray crystallography. The gene encoding ScEst was cloned into the pVFT1S vector and overexpressed in E. coli. The recombinant ScEst protein was obtained by affinity and size-exclusion purification. ScEst showed substrate preference for the short chain length of acyl derivatives. It was crystallized in an optimized solution composed of 0.25 M ammonium citrate tribasic (pH 7.0) and 20% PEG 3350 at 296 K. A total of 360 X-ray diffraction images were collected at a 1.66 Å resolution. ScEst crystal belongs to the space group of P212121 with the unit cell parameters of a = 50.23 Å, b = 68.69 Å, c = 71.15 Å, and α = β = γ = 90°. Structure refinement after molecular replacement is under progress. Further biochemical studies will elucidate the hydrolysis mechanism of ScEst. Overall, this study is the first to report the functional characterization of an esterase from Staphylococcus chromogenes, which is potentially useful in elaborating its hydrolysis mechanism.

Identification, Characterization, and Preliminary X-ray Diffraction Analysis of a Single Stranded DNA Binding Protein (LjSSB) from Psychrophilic Lacinutrix jangbogonensis PAMC 27137

Single-stranded DNA-binding proteins (SSBs) are essential for DNA metabolism, including repair and replication, in all organisms. SSBs have potential applications in molecular biology and in analytical methods. In this study, for the first time, we purified, structurally characterized, and analyzed psychrophilic SSB (LjSSB) from Lacinutrix jangbogonensis PAMC 27137 isolated from the Antarctic region. LjSSB has a relatively short amino acid sequence, consisting of 111 residues, with a molecular mass of 12.6 kDa. LjSSB protein was overexpressed in Escherichia coli BL21 (DE3) and analyzed for binding affinity using 20- and 35-mer deoxythymidine oligonucleotides (dT). In addition, the crystal structure of LjSSB at a resolution 2.6 Å was obtained. The LjSSB protein crystal belongs to the space group C222 with the unit cell parameters of a = 106.58 Å, b = 234.14 Å, c = 66.14 Å. The crystal structure was solved using molecular replacement, and subsequent iterative structure refinements and model building are currently under progress. Further, the complete structural information of LjSSB will provide a novel strategy for protein engineering and for the application on molecular biological techniques.

Purification, crystallization, and preliminary X-ray diffraction analysis of an S-formylglutathione hydrolase (VaSFGH) homolog from Variovorax sp. PAMC 28711

Purification, crystallization, and preliminary X-ray diffraction analysis of an <i>S</i>-formylglutathione hydrolase (<i>Va</i>SFGH) homolog from <i>Variovorax</i> sp. PAMC 28711

Purification, Crystallization, and Preliminary Crystallographic Studies of Human As(III) S-Adenosylmethionine Methyltransferase (hAS3MT).

Exposure to environmental arsenic is associated with serious of health issues such as cancer, diabetes and developmental delays in infants and children. In human liver, As(III) S-adenosylmethionine methyl transferase (hAS3MT) (EC 2.1.1.137) was proposed to be an detoxification process by methylation of inorganic arsenite into pentavalent methyl MAs(V) and dimethyl arsenite DMAs(V). More recently the first product was shown to be highly toxic and potentially carcinogenic trivalent methylarsenite (MAs(III)). Our studies are designed to elucidate the mechanism of AS3MT and its contribution to arsenic-related diseases. Here, we report the first crystallization and preliminary X-ray diffraction analysis of the human AS3MT enzyme. The crystals belong to the monoclinic P1211 space group with unit cell parameters of a = 135.03 Å, b = 260.44 Å, c = 279.03 Å, α = 90.00°, β = 93.36°, γ = 90.00°.

ランタニド金属含有アルコール脱水素酵素の生理的電子受容体シトクロム c553 の結晶化と予備的X線回折実験

ランタニド金属含有アルコール脱水素酵素の生理的電子受容体シトクロム c553 の結晶化と予備的X線回折実験

Crystallization and Preliminary X-Ray Diffraction Analysis of the ZAD Domain of the Serendipity-d Protein from Drosophila melanogaster

The spatial organization of the genome is controlled by a special class of architectural proteins, in particular proteins containing ZAD domains that are able to form homo- and heterodimers. These domains are unique for arthropods and are present at the N-terminus of many proteins containing C2H2-type zinc fingers. Despite a growing interest in the characterization of architectural proteins, little is known about their structures (structures of their functional domains), which could shed light on the mechanisms of regulatory processes of the establishment and maintenance of the spatial organization of chromatin domains. Only two structures of ZAD domains are known. Here we report the crystallization and preliminary X-ray diffraction analysis of the previously unknown ZAD domain of the Serendipity-d protein from Drosophila melanogaster.

Adsorption Properties of Ce5(BDC)7.5(DMF)4 MOF

In this article we report on the spectroscopic and adsorptive studies done on Ce(III)-based MOF possessing, upon desolvation, open metal sites, and a discrete surface area. The Ce-based MOF was synthesized from terephthalic acid linker (H2BDC) and Ce3+ cations by the classical solvothermal method. Preliminary powder X-ray diffraction analysis showed that the obtained materials corresponded to the ones reported by other authors. Spectroscopic techniques, such as XAS and in situ FTIR with probe molecules were used. In situ FTIR spectroscopy confirmed the successful removal of DMF molecules within the pore system at temperatures above 250 °C. Moreover, the use of CO as a probe molecule evidenced the presence of a Ce3+ open metal sites. Detailed volumetric and calorimetric CO2 adsorption studies are also reported.

Crystallization and Preliminary X-ray diffraction Analysis of the Anammox Copper Nitrite Reductase

Crystallization and Preliminary X-ray diffraction Analysis of the Anammox Copper Nitrite Reductase

Crystallization and preliminary X-ray diffraction analysis of Thioredoxin from the feather-degrading thermophile Fervidobacterium islandicum AW-1

Crystallization and preliminary X-ray diffraction analysis of Thioredoxin from the feather-degrading thermophile Fervidobacterium islandicum AW-1

Expression, purification, crystallization and preliminary X-ray diffraction analysis of swine leukocyte antigen 2 complexed with a CTL epitope AS64 derived from Asia1 serotype of foot-and-mouth disease virus

BackgroundCurrently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified.ResultsA complex of SLA-I constituted by an SLA-2*HB01 molecule with swine β2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64–72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å.ConclusionThis research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.

BpeB, a major resistance-nodulation-cell division transporter from Burkholderia cenocepacia: construct design, crystallization and preliminary structural analysis.

Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to remove possible disordered regions based on comparative sequence and structural analysis to salvage the wild-type protein, which failed to crystallize. The 17-residue carboxyl-terminal truncation yielded crystals that diffracted to 3.6 Å resolution. The efflux function measured using minimal inhibitory concentration assays indicated that the truncation decreased, but did not eliminate, the efflux activity of the transporter.

Crystallization and preliminary X-ray diffraction analysis of YejM from Salmonella typhimurium: an essential inner membrane protein involved in outer membrane directed cardiolipin transport.

Salmonella typhimuriumis responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacteriumpossesses an inner and an outer membrane (OM), the latter allowingits survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Successful structure determination of YejM will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately, yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.

Crystallization and preliminary X-ray diffraction analysis of YejM from Salmonella typhimurium: an essential inner membrane protein involved in outer membrane directed cardiolipin transport

Salmonella typhimurium is responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacterium possesses an inner and an outer membrane (OM), the latter allowing its survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Successful structure determination of YejM will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately, yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.

Dengue virus 3 NS5 methyltransferase domain: expression, purification, crystallization and first structural data from microcrystalline specimens

Abstract Flavivirus infections often provoke life-threatening diseases of epidemic magnitudes, thus extensive research is currently directed towards the development of efficient vaccines and approved antiviral compounds. We present here the expression, purification, crystallization and preliminary X-ray diffraction analysis of one of the components of the flavivirus replication complex, the non-structural protein 5 (NS5) mRNA methyltransferase (MTase) domain, from an emerging pathogenic flavivirus, dengue virus 3 (DEN3). Polycrystalline precipitates of DEN3 NS5 MTase, suitable for X-ray powder diffraction (XRPD) measurements, were produced in the presence of PEG 8000 (25–32.5% (w/v)), 0.1 M Tris-Amino, in a pH range from 7.0 to 8.0. A polymorph of orthorhombic symmetry (space group: P21212, a=61.9 Å, b=189.6 Å, c=52.4 Å) was identified via XRPD. These results are the first step towards the complete structural determination of this molecule via XRPD and a parallel demonstration of the applicability of the method.

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°С, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-A resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 A, b = 112.6 A, c = 110.2 A, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-A resolution.

Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights.

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl2 in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a=77.9, b = 65.7, c=40.6 Å, β = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.