Preliminary X-ray Diffraction Analysis Research Articles

Preliminary X-ray diffraction analysis of a thermophilic β-1,3-1,4-glucanase from Clostridium thermocellum.

β-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a=b=74.5, c=182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.

Crystallization and preliminary X-ray diffraction analysis of Gos1p, a yeast SNARE protein.

The Gos1 protein (Golgi SNAP receptor complex member 1) is involved in the SNARE complex, which is the core machinery that drives membrane fusion between cargo-carrying vesicles and their target membranes in the secretory and endocytic pathways in yeast. Truncated versions of the Gos1 protein from Saccharomyces cerevisiae were cloned, expressed, purified and crystallized. The crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a=39.67, b=43.58, c=81.94 Å, α=β=γ=90°. An X-ray diffraction data set was collected at 100 K to 1.63 Å resolution. Matthews coefficient (VM) calculations suggest that one molecule is present in the asymmetric unit, corresponding to a solvent content of ∼55%.

Purification, crystallization and preliminary X-ray diffraction analysis of Imp3 in complex with an Mpp10 peptide involved in yeast ribosome biogenesis.

Eukaryotic ribosome synthesis requires a vast number of transiently associated factors. Mpp10, Imp3 and Imp4 form a protein complex in the 90S pre-ribosomal particle that conducts early processing of 18S rRNA. Here, a short fragment of Mpp10 was identified to associate with and increase the solubility of Imp3. An Imp3-Mpp10 complex was co-expressed, co-purified and co-crystallized. Preliminary X-ray diffraction analysis revealed that the crystal diffracted to 2.1 Å resolution and belonged to space group P2₁2₁2₁, with unit-cell parameters a=51.6, b=86.9, c=88.7 Å.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Streptococcus agalactiae NEM316.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme involved in glycolysis. Despite lacking the secretory signal sequence, this cytosolic enzyme has been found localized at the surface of several bacteria and fungi. As a surface protein, GAPDH exhibits various adhesive functions, thereby facilitating colonization and invasion of host tissues. Streptococcus agalactiae, also known as group B streptococcus (GBS), binds onto the host using its surface adhesins and causes sepsis and pneumonia in neonates. GAPDH is one of the surface adhesins of GBS binding to human plasminogen and is a virulent factor associated with host colonization. Although the surface-associated GAPDH has been shown to bind to a variety of host extracellular matrix (ECM) molecules in various bacteria, the molecular mechanism underlying their interaction is not fully understood. To investigate this, structural studies on GAPDH of S. agalactiae were initiated. The gapC gene of S. agalactiae NEM316 encoding GAPDH protein was cloned into pET-28a vector, overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity. The purified protein was crystallized using the hanging-drop vapour-diffusion method. The GAPDH crystals obtained in two different crystallization conditions diffracted to 2.8 and 2.6 Å resolution, belonging to two different space groups P2₁ and P2₁2₁2₁, respectively. The structure was solved by molecular replacement and structure refinement is now in progress.

Crystallization and preliminary X-ray diffraction analysis of (R)-carbonyl reductase from Candida parapsilosis.

The NADH-dependent (R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce (R)-1-phenyl-1,2-ethanediol [(R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=85.64, b=106.11, c=145.55 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.15 Å resolution. Initial model building indicates that RCR forms a homotetramer, consistent with previous reports of medium-chain-type alcohol dehydrogenases.

Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system inThermotoga maritima

A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3₁12 (or P3₂12), with unit-cell parameters a=b=88.9, c=221.2 Å, α=90, β=90, γ=120°, indicating that a dimer was present in the asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase from Streptomyces sp. NRRL 2288.

AntE from Streptomyces sp. NRRL 2288 is a crotonyl-CoA carboxylase/reductase that catalyzes the reductive carboxylation of various α,β-unsaturated acyl-CoAs to provide the building block at the C7 position for antimycin A biosynthesis. Recombinant AntE expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group I222 or I2₁2₁2₁, with unit-cell parameters a=76.4, b=96.7, c=129.6 Å, α=β=γ=90.0°. A diffraction data set was collected at the KEK Photon Factory to 2.29 Å resolution.

Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms.

Cel7A (previously known as cellobiohydrolase I) from Hypocrea jecorina was crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space group C2, with unit-cell parameters a=152.5, b=44.9, c=57.6 Å, β=101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space group P6₃22, with unit-cell parameters a=b≃155, c≃138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.

Crystallization and preliminary X-ray diffraction analysis of proximal thread matrix protein 1 (PTMP1) from Mytilus galloprovincialis.

In order to deal with the dynamic ocean environment, blue mussels adhere to various surfaces via their collagenous byssal threads. PTMP1 (proximal thread matrix protein 1) is one identified matrix protein residing in the proximal thread and is capable of collagen binding. Its sequence comprises two von Willebrand factor type A-like repeats. In order to characterize the structure and domain architecture of PTMP1, recombinant protein was crystallized by vapour diffusion. The obtained crystals diffracted to 1.95 Å resolution and belonged to space group P2₁, with unit-cell parameters a=62.0, b=62.3, c=122.6 Å, β=102.2°. The Matthews coefficient suggested the presence of two monomers in the asymmetric unit and 48.3% solvent content.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal NHL domain of human TRIM2.

The tripartite motif-containing protein 2 (TRIM2) functions as an E3 ubiquitin ligase. Loss of function of TRIM2 has been shown to result in early-onset axonal neuropathy. As a member of the TRIM-NHL family of proteins, TRIM2 has a conserved modular architecture that includes N-terminal RING finger and B-box domains, a middle coiled-coil domain and a C-terminal NHL domain. To characterize the functional role of its NHL domain from the perspective of structural biology, a truncation of human TRIM2 (residues 465-744) was expressed, purified and crystallized. Rod-shaped crystals were obtained that diffracted X-rays to 1.7 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 43.6, b = 76.4, c = 107.4 Å, α = 90.0, β = 94.0, γ = 90.0°. A Matthews coefficient of 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.6%, indicated the presence of three molecules per asymmetric unit, which was further confirmed by the phasing solution from molecular replacement.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan.

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

Crystallization and preliminary X-ray diffraction analysis of a novel β-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum.

The β-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3x21, with unit-cell parameters a = b = 75.9, c = 254.0Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78Å resolution. A BLASTP search (http://blast.ncbi.nlm.nih.gov/) of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress.

Crystallization and preliminary X-ray crystallographic analysis of importin-α from Neurospora crassa.

Importin-α recognizes cargo proteins that contain classical nuclear localization sequences (NLS) and, in complex with importin-β, is able to translocate nuclear proteins through the nuclear pore complex. The filamentous fungus Neurospora crassa is a well studied organism that has been widely used as a model organism for fundamental aspects of eukaryotic biology, and is important for understanding the specific mechanisms of protein transport to the cell nucleus. In this work, the crystallization and preliminary X-ray diffraction analysis of importin-α from N. crassa (IMPα-Nc) complexed with a classical NLS peptide (SV40 NLS) are reported. IMPα-Nc-SV40 NLS crystals diffracted X-rays to 2.0 Å resolution and the structure was solved by molecular-replacement techniques, leading to a monomeric structure. The observation of the electron-density map indicated the presence of SV40 NLSs interacting at both the minor and major NLS-binding sites of the protein.

Purification, crystallization and preliminary X-ray diffraction analysis of ArsH from Synechocystis sp. strain PCC 6803.

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of an ars operon. The function of the arsH gene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH from Synechocystis sp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space group I4122, with unit-cell parameters a = b = 127.94, c = 65.86 Å and one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH from Sinorhizobium meliloti will provide information about the oligomerization of ArsH.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.

The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.

Cloning, crystallization and preliminary X-ray diffraction analysis of an intact DNA methyltransferase of a type I restriction-modification enzyme from Vibrio vulnificus.

Independently of the restriction (HsdR) subunit, the specificity (HsdS) and methylation (HsdM) subunits interact with each other, and function as a methyltransferase in type I restriction-modification systems. A single gene that combines the HsdS and HsdM subunits in Vibrio vulnificus YJ016 was expressed and purified. A crystal suitable for X-ray diffraction was obtained from 25%(w/v) polyethylene glycol monomethylether 5000, 0.1 M HEPES pH 8.0, 0.2 M ammonium sulfate at 291 K by hanging-drop vapour diffusion. Diffraction data were collected to a resolution of 2.31 Å using synchrotron radiation. The crystal belonged to the primitive monoclinic space group P21, with unit-cell parameters a = 93.25, b = 133.04, c = 121.49 Å, β = 109.7°. With four molecules in the asymmetric unit, the crystal volume per unit protein weight was 2.61 Å(3) Da(-1), corresponding to a solvent content of 53%.

Structural and electrical properties of La-modified BiFeO3–BaTiO3 composites

In the present investigation, La-modified solid solutions of BiFeO3 (BFO) and BaTiO3 (BT) in different molar ratios [i.e., (Bi0.5−x La x Ba0.5)(Fe0.5Ti0.5)O3, with x = 0.0, 0.05, 0.10 and 0.15)] have been synthesized using a solid-state reaction route. Structural and electrical properties of single phase (with minor secondary phase) of BFO–BT system have been studied in details to understand their ferroelectric and other properties. Preliminary X-ray diffraction analysis confirms the formation of a new system, which is different from that of its parent compounds. Substitution of a small amount BaTiO3 into BiFeO3 enhances dielectric and ferroelectric responses and reduces electrical leakage or tangent loss. The ac conductivity obeys Jonscher’s universal power law. The electrical behavior of the samples was investigated by impedance spectroscopy in a wide temperature range (25–525 °C) at different frequency (1 kHz–1 MHz). The impedance spectroscopy of the materials also confirms the origin of the relaxation mechanism in the system.

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Crystallization and preliminary X-ray diffraction analysis of the Fab portion of the Alzheimer's disease immunotherapy candidate bapineuzumab complexed with amyloid-β.

Bapineuzumab (AAB-001) and its derivative (AAB-003) are humanized versions of the anti-Aβ murine antibody 3D6 and are immunotherapy candidates in Alzheimer's disease. The common Fab fragment of these immunotherapies has been expressed, purified and crystallized in complex with β-amyloid peptides (residues 1-8 and 1-28). Diffraction data at high resolution were acquired from crystals of Fab-Aβ8 (2.0 Å) and Fab-Aβ28 (2.2 Å) complexes at the Australian Synchrotron. Both crystal forms belonged to the primitive orthorhombic space group P21221.

Crystallization and preliminary X-ray diffraction analysis of the interleukin-3 alpha receptor bound to the Fab fragment of antibody CSL362.

Interleukin-3 (IL-3) is a member of the beta common family of cytokines that regulate multiple functions of myeloid cells. The IL-3 receptor-specific alpha subunit (IL3Rα) is overexpressed on stem cells/progenitor cells of patients with acute myeloid leukaemia, where elevated receptor expression correlates clinically with a reduced patient survival rate. The monoclonal antibody (MAb) CSL362 is a humanized MAb derived from the murine MAb 7G3, originally identified for its ability to specifically recognize the human IL-3 receptor and for blocking the signalling of IL-3 in myeloid and endothelial cells. In order to elucidate the molecular mechanism of CSL362 antagonism, a preliminary structure of human IL3Rα in complex with the MAb CSL362 has been determined.