Preliminary X-ray Diffraction Analysis Research Articles

Sequence similarity network analysis, crystallization, and X-ray crystallographic analysis of the lactate metabolism regulator LldR from Pseudomonas aeruginosa

The FadR subfamily of regulators plays essential roles in the regulation of diverse metabolic pathways in bacteria. LldR, an FadR-type regulator, regulates lactate utilization in Pseudomonas aeruginosa. Sequence network analysis of the LldR proteins from different bacterial species showed that LldR proteins from Pseudomonas sp. and Escherichia coli were separated into different clusters, suggesting that LldRs are derived from two ancestors that functionally diverged. Then, the recombinant PLldR protein (LldR of P. aeruginosa) was expressed, purified, and crystallized. Preliminary X-ray diffraction analysis of LldR protein crystals was performed. The PLldR crystal diffracted to 2.55 A resolution and belonged to the trigonal space group P3, with unit-cell parameters a = 68.5 A, b = 68.5 A, and c = 237.0 A. These results will facilitate further understanding of the regulatory mechanism and the adaptation to sensing of both l -lactate and d -lactate of LldR proteins from Pseudomonas sp. in lactate metabolism.

Electrical and ferroelectric characteristics of (LaLi)1/2(Fe2/3Mo1/3)O3

Using high-temperature solid state reaction technique a new polycrystalline multiferroic material of (LaLi)1/2(Fe2/3Mo1/3)O3 has been prepared. Thermo-gravimetric analysis is carried out to optimize the calcination temperature of the material. The formation of the desired compound is confirmed by preliminary X-ray diffraction analysis. The complex impedance spectroscopy technique is used to study the dielectric properties and other electrical parameters (loss tangent, impedance, modulus, conductivity) of the material as a function of frequency (1 kHz–1 MHz) at different temperatures (RT−425 °C). The nature of Nyquist plot confirmed the presence of bulk and grain boundary effect in the compound. The relaxation time obtained from the maximum frequencies corresponding to the imaginary impedance and electric modulus plots at different temperatures are found to obey Arrhenius law. The temperature and frequency dependence of ac conductivity plot obeys Jonscher’s universal power law. The P–E loop confirms the ferroelectric behavior of the compound at room temperature.

Swinging Symmetry, Multiple Structural Phase Transitions, and Versatile Physical Properties in RECuGa3 (RE = La-Nd, Sm-Gd).

The compounds RECuGa3 (RE = La-Nd, Sm-Gd) were synthesized by various techniques. Preliminary X-ray diffraction (XRD) analyses at room temperature suggested that the compounds crystallize in the tetragonal system with either the centrosymmetric space group I4/mmm (BaAl4 type) or the non-centrosymmetric space group I4mm (BaNiSn3 type). Detailed single-crystal XRD, neutron diffraction, and synchrotron XRD studies of selected compounds confirmed the non-centrosymmetric BaNiSn3 structure type at room temperature with space group I4mm. Temperature-dependent single-crystal XRD, powder XRD, and synchrotron beamline measurements showed a structural transition between centro- and non-centrosymmetry followed by a phase transition to the Rb5Hg19 type (space group I4/m) above 400 K and another transition to the Cu3Au structure type (space group Pm3̅m) above 700 K. Combined single-crystal and synchrotron powder XRD studies of PrCuGa3 at high temperatures revealed structural transitions at higher temperatures, highlighting the closeness of the BaNiSn3 structure to other structure types not known to the RECuGa3 family. The crystal structure of RECuGa3 is composed of eight capped hexagonal prism cages [RE4Cu4Ga12] occupying one rare-earth atom in each ring, which are shared through the edge of Cu and Ga atoms along the ab plane, resulting in a three-dimensional network. Resistivity and magnetization measurements demonstrated that all of these compounds undergo magnetic ordering at temperatures between 1.8 and 80 K, apart from the Pr and La compounds: the former remains paramagnetic down to 0.3 K, while superconductivity was observed in the La compound at T = 1 K. It is not clear whether this is intrinsic or due to filamentary Ga present in the sample. The divalent nature of Eu in EuCuGa3 was confirmed by magnetization measurements and X-ray absorption near edge spectroscopy and is further supported by the crystal structure analysis.

Expression, Crystallization and Preliminary X-ray Diffraction Analyses of Med-ORF10 in the Biosynthetic Pathway of an Antitumor Antibiotic Medermycin.

Medermycin, as a prominent member of benzoisochromanequinones, possesses strong antitumor activity and is biosynthesized under the control of a 29-ORF-containing biosynthetic gene cluster. Most of ORFs in this gene cluster have not been characterized, including a small protein encoding gene med-ORF10, proposed to play a regulatory role in biosynthesis of medermycin in an unknown mode. In this study, we reported the expression, protein preparation, crystallization and preliminary X-ray diffraction analyses of Med-ORF10 of the wild type Streptomyces strain. Firstly, we cloned and overexpressed med-ORF10 in Escherichia coli and purified the protein with 98% purity and 3 mg/L yield. Then, we crystallized the protein at concentration of 20 mg/mL in condition 22% PEG 3350, 0.2 M magnesium formate and collected the data at 1.78 Å resolution. Finally, we detected the expression of Med-ORF10 in Streptomyces by western blotting. In conclusion, this study confirmed the expression of Med-ORF10 protein in the wild-type strain of Streptomyces AM-7161 and collected the X-ray diffraction data of Med-ORF10 crystal at 1.78 Å resolution. These studies provide evidences for the functional Med-ORF10 protein in Streptomyces strains and facilitate our further investigation.

The effects of lateral halogen substituents on the low-temperature cybotactic nematic phase in oxadiazole based bent-core liquid crystals

We have previously demonstrated that the incorporation of lateral methyl groups on oxadiazole-based liquid crystals leads to relatively low-temperature cybotactic nematic phases which, in some cases, supercool to room temperature. We report here the synthesis and phase behaviour of related compounds that possess lateral halogen groups and in some cases, lateral methyl groups as well. Derivatives with three lateral substituents (one halogen and two methyl groups) in a specific pattern supercool in the nematic phase to room temperature. As was the case with the previously reported trimethylated derivatives, the low-temperature nematic phase is glassy in nature. Two of the new trisubstituted derivatives (with bromo and chloro groups) remain in the nematic phase upon subsequent heating until transitioning to the isotropic phase indicating that the low-temperature nematic phase may be more stable than that shown by the trimethylated analogue. Preliminary X-ray diffraction analysis confirms the presence of a tilted cybotactic nematic phase. In addition, the splitting observed in the wide-angle scattering feature is indicative of enhanced local biaxial packing.

Purification, crystallization, crystallographic analysis and phasing of the CRISPR-associated protein Csm2 from Thermotoga maritima.

The clusters of regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system consists of an intriguing machinery of proteins that confer bacteria and archaea with immunity against phages and plasmids via an RNA-guided interference mechanism. Here, the cloning, recombinant expression in Escherichia coli BL21 (DE3), purification, crystallization and preliminary X-ray diffraction analysis of Csm2 from Thermotoga maritima are reported. Csm2 is thought to be a component of an important protein complex of the type IIIA CRISPR-Cas system, which is involved in the CRISPR-Cas RNA-guided interference pathway. The structure of Csm2 was solved via cadmium single-wavelength anomalous diffraction (Cd-SAD) phasing. Owing to its involvement in the CRISPR-Cas system, the crystal structure of this protein could be of importance in elucidating the mechanism of type IIIA CRISPR-Cas systems in bacteria and archaea.

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds.

Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b=74.56, c= 118.98 Å, β = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

Purification, crystallization and preliminary X-ray diffraction analysis of 3-ketoacyl-CoA thiolase A1887 from Ralstonia eutropha H16.

The gene product of A1887 from Ralstonia 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 purified ReH16_A1887 was measured and enzyme-kinetic parameters for the protein were obtained, with Km, Vmax and kcat values of 158 µM, 32 mM min(-1) and 5 × 10(6) s(-1), respectively. The ReH16_A1887 protein was crystallized in 17% PEG 8K, 0.1 M HEPES pH 7.0 at 293 K and a complete data set was collected to 1.4 Å resolution. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 129.52, c = 114.13 Å, α = β = γ = 90°. The asymmetric unit contained two molecules, with a solvent content of 58.9%.

Crystallization and preliminary X-ray diffraction analysis of the two distinct types of zebrafish β2-microglobulin.

β(2)-Microglobulin (β(2)m) noncovalently associates with the heavy chain of major histocompatibility complex class I (MHC I) molecules, which bind foreign antigen peptides to control the cytotoxic T lymphocyte (CTL) immune response. In contrast to mammals, there are distinct types of β(2)ms derived from two loci in a number of teleost species. In order to clarify the structures of the β(2)ms, the zebrafish (Danio rerio) β(2)ms Dare-β(2)m-I and Dare-β(2)m-II were expressed in Escherichia coli, purified and crystallized, and diffraction data were collected to 1.6 and 1.9 Å resolution, respectively. Both crystals belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a = 38.2, b = 50.4, c = 50.9 Å for Dare-β(2)m-I and a = 38.9, b = 52.7, c = 65.8 Å for Dare-β(2)m-II. Each asymmetric unit was constituted of one molecule, with Matthews coefficients of 2.22 and 3.01 Å(3) Da(-1) and solvent contents of 45 and 59% for Dare-β(2)m-I and Dare-β(2)m-II, respectively. These two β(2)m structures will provide relevant information for further studies of the structures of the MHC I complex.

Crystallization and preliminary X-ray diffraction analysis of the CRISPR-Cas RNA-silencing Cmr complex.

Clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNA (crRNA) and CRISPR-associated (Cas) proteins constitute a prokaryotic adaptive immune system (CRISPR-Cas system) that targets and degrades invading genetic elements. The type III-B CRISPR-Cas Cmr complex, composed of the six Cas proteins (Cmr1-Cmr6) and a crRNA, captures and cleaves RNA complementary to the crRNA guide sequence. Here, a Cmr1-deficient functional Cmr (CmrΔ1) complex composed of Pyrococcus furiosus Cmr2-Cmr3, Archaeoglobus fulgidus Cmr4-Cmr5-Cmr6 and the 39-mer P. furiosus 7.01-crRNA was prepared. The CmrΔ1 complex was cocrystallized with single-stranded DNA (ssDNA) complementary to the crRNA guide by the vapour-diffusion method. The crystals diffracted to 2.1 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 75.5, b = 76.2, c = 139.2 Å, α = 90.3, β = 104.8, γ = 118.6°. The asymmetric unit of the crystals is expected to contain one CmrΔ1-ssDNA complex, with a Matthews coefficient of 2.03 Å(3) Da(-1) and a solvent content of 39.5%.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus.

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.8, b = 86.0, c = 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-replacement solution was obtained and model building and refinement are currently under way.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis.

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF from Microcystis aeruginosa with a C-terminal His6 tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space group P4322, with unit-cell parameters a = b = 101.581, c = 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å(3) Da(-1) and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor ArgR from Bacillus halodurans.

The arginine repressor (ArgR) is a transcriptional regulator which regulates genes encoding proteins involved in arginine biosynthesis and the arginine catabolic pathway. ArgR from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. ArgR (Bh2777) from B. halodurans is composed of 149 amino-acid residues with a molecular mass of 16 836 Da. ArgR was crystallized at 296 K using 1,2-propanediol as a precipitant. Crystals of N-terminally His-tagged ArgR were obtained by the sitting-drop vapour-diffusion method. Dehydrated crystals showed a dramatic improvement in diffraction quality and diffracted to 2.35 Å resolution. The crystals belonged to the cubic space group I23, with unit-cell parameters a = b = c = 104.68 Å. The asymmetric unit contained one monomer of ArgR, which generates a trimer by the threefold axis of the space group, giving a crystal volume per mass (VM) of 2.98 Å(3) Da(-1) and a solvent content of 56.8%.

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of Haemophilus influenzae BamD and BamCD complex.

The Bam machinery, which is highly conserved from bacteria to humans, is well recognized as the apparatus responsible for the insertion and folding of most outer membrane proteins in Gram-negative bacteria. In Escherichia coli, the Bam machinery consists of five components (i.e. BamA, BamB, BamC, BamD and BamE). In comparison, there are only four partners in Haemophilus influenzae: a BamB homologue is not found in its genome. In this study, the recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of H. influenzae BamD and BamCD complex are reported. The genes encoding BamC and BamD were cloned into a pET vector and expressed in E. coli. Affinity, ion-exchange and gel-filtration chromatography were used to obtain high-purity protein for further crystallographic characterization. Using the hanging-drop vapour-diffusion technique, BamD and BamCD protein crystals of suitable size were obtained using protein concentrations of 70 and 50 mg ml(-1), respectively. Preliminary X-ray diffraction analysis showed that the BamD crystals diffracted to 4.0 Å resolution and belonged to space group P212121, with unit-cell parameters a = 54.5, b = 130.5, c = 154.7 Å. The BamCD crystals diffracted to 3.8 Å resolution and belonged to space group I212121, with unit-cell parameters a = 101.6, b = 114.1, c = 234.9 Å.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the effector-interaction domain of the resistance protein RGA5-A from Oryza sativa L. japonica.

RGA5-A, a component of the Pia resistance-protein complex (RGA4/RGA5-A) from Oryza sativa L. japonica, has the ability to interact physically with the effector protein AVR-Pia from Magnaporthe oryzae via its effector-interaction domain RGA5-A_S. The interaction between RGA5-A and AVR-Pia relieves the repression of RGA4, leading to AVR-independent cell death by the freed RGA4. To further understand the details of this interaction, the effector-interaction domain RGA5-A_S was expressed in Escherichia coli and purified to homogeneity. The purified recombinant protein His-RGA5-A_S was successfully crystallized using the sitting-drop vapour-diffusion method. A single crystal obtained using 0.2 M ammonium citrate, 25%(w/v) PEG 3350 diffracted to 2.43 Å resolution. It belonged to space group P4122 or P4322, with unit-cell parameters a = b = 55.2, c = 78.2 Å, α = β = γ = 90°.

Crystallization and preliminary X-ray diffraction analysis of a putative carbon-carbon bond hydrolase from Mycobacterium abscessus 103.

The PhlG protein from Mycobacterium abscessus 103 (mPhlG), which shares 30% sequence identity with phloretin hydrolase from Eubacterium ramulus and 38% sequence identity with 2,4-diacetylphloroglucinol hydrolase from Pseudomonas fluorescens Pf-5, is a putative carbon-carbon bond hydrolase. Here, the expression, purification and crystallization of mPhlG are reported. Crystals were obtained using a precipitant consisting of 100 mM citric acid pH 5.0, 1.0 M lithium chloride, 8%(w/v) polyethylene glycol 6000. The crystals diffracted to 1.87 Å resolution and belonged to space group P21, with unit-cell parameters a = 71.0, b = 63.4, c = 74.7 Å, α = 90.0, β = 103.2, γ = 90.0°. Assuming the presence of two mPhlG molecules in the asymmetric unit, VM was calculated to be 2.5 Å(3) Da(-1), which corresponds to a solvent content of 50%.

Preliminary X-ray analysis of the binding domain of the soybean vacuolar sorting receptor complexed with a sorting determinant of a seed storage protein.

β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α' and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α' and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 Å.

Crystallization and preliminary X-ray diffraction studies of the family 54 carbohydrate-binding module from laminarinase (β-1,3-glucanase) Lic16A of Clostridium thermocellum.

The crystallization and preliminary X-ray diffraction analysis of the carbohydrate-binding module (CBM) from laminarinase Lic16A of the hyperthermophilic anaerobic bacterium Clostridium thermocellum (ctCBM54) are reported. Recombinant ctCBM54 was prepared using an Escherichia coli/pQE30 overexpression system and was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystals belonged to space group P6322, with unit-cell parameters a = b = 130.15, c = 131.05 Å. The three-dimensional structure of ctCBM54 will provide valuable information about the structure-function relation of the laminarinase Lic16A and will allow the exploitation of this binding module in biotechnological applications.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of SF173 from Shigella flexneri.

Shigella flexneri is a Gram-negative, anaerobic bacterium in the genus Shigella that can cause diarrhoea in humans. SF173, a hypothetical protein from S. flexneri 5a strain M90T, has been cloned, overexpressed, purified and crystallized as a part of laboratory-scale structural genomics project. The SF173 protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 0.8 M succinic acid pH 7.0 at 293 K. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 1.47 Å resolution and belonged to space group I432, with unit-cell parameters a=b=c=110.245 Å.

Crystallization and preliminary X-ray diffraction analysis of Trap1 complexed with Hsp90 inhibitors.

Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293 K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1-PU-H71 (2.7 Å) and Trap1-BIIB-021 (3.1 Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 69.2, c = 252.5 Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.