Acta Crystallographica Section F-structural Biology and Crystallization Communications最新文献

Avian infectious bronchitis virus (IBV) is a member of the group III coronaviruses, which differ from the other groups of coronaviruses in that they do not encode the essential pathogenic factor nonstructural protein 1 (nsp1) and instead start with nsp2. IBV nsp2 is one of the first replicase proteins to be translated and processed in the viral life cycle; however, it has an entirely unknown function. In order to better understand the structural details and functional mechanism of IBV nsp2, the recombinant protein was cloned, overexpressed in Escherichia coli, purified and crystallized. The crystals diffracted to 2.8 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 57.0, b = 192.3, c = 105.7 Å, β = 90.8°. Two molecules were found in the asymmetric unit; the Matthews coefficient was 3.9 Å(3) Da(-1), corresponding to a solvent content of 68.2%.

The LysR-type transcriptional regulator MvfR plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing-regulated virulence factors. The protein also controls pathogenic type VI secretion loci. MvfRC87, a 242-residue C-terminal segment of MvfR, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected using synchrotron radiation and crystallographic parameters were determined.

Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-L-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, β = 96.93°.

The CheA-CheW complex plays a key role in bacterial chemotaxis signal transduction by initiating phosphotransfer to response regulators via coupling to the chemoreceptors. CheA (P3-P4-P5 domains) and CheW from Thermotoga maritima were overexpressed in Escherichia coli and crystallized as a complex at 298 K using ammonium dihydrogen phosphate as a precipitant. X-ray diffraction data were collected to ~8 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 184.2, b = 286.4, c = 327.7 Å. The asymmetric unit may contain six to ten CheA-CheW molecules.

Soluble forms of recombinant LukE protein (expressed in Escherichia coli) and of wild-type LukD protein (expressed in Staphylococcus aureus), which together form the staphylococcal LukE-LukD leukotoxin, were purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The crystals of LukE belonged to space group I4, with unit-cell parameters a = b = 134.50, c = 64.43 Å, and diffracted X-rays to 1.6 Å resolution. The crystals of LukD belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.04, b = 50.99, c = 137.40 Å, and diffracted to 1.9 Å resolution. Molecular replacement using the LukF-PV structure (PDB entry 1pvl) as a template model allowed the identification of an initial structure solution for the LukD data. In the case of LukE, a solution comprising only a single copy of the search model (LukS-PV; PDB entry 1t5r) was found, although the unit-cell parameters indicated that up to three molecules could be accommodated in the asymmetric unit.

In the presence of calcium ions, human peptidylarginine deiminase (PAD) converts arginine residues in proteins to citrulline. Of the five known human PAD enzymes, the type III isozyme (PAD3) exhibits the highest specificity for synthetic and natural substrates. This study aimed to determine the structure of PAD3 in order to elucidate its selective citrullination mechanism. Crystals of PAD3 obtained using polyethylene glycol 400 as a precipitant diffracted to 2.95 Å resolution using synchrotron radiation. They belonged to space group R3, with unit-cell parameters a = b = 114.97, c = 332.49 Å (hexagonal axes). Assuming two molecules were contained in an asymmetric unit, the calculated Matthews coefficient was 2.83 Å(3) Da(-1), corresponding to a solvent content of 56.6%. Initial phases were determined using PAD4 as a molecular-replacement model.

The human transporter associated with antigen processing (TAP) protein belongs to the ATP-binding cassette (ABC) transporter superfamily and is formed by the heterodimerization of TAP1 and TAP2 subunits. TAP selectively pumps cytosolic peptides into the lumen of the endoplasmic reticulum in an ATP-dependent manner. The catalytic cycle of the ATPase domain of TAP is not understood at the molecular level. The structures of catalytic intermediates of the ATPase domain of TAP will contribute to the understanding of the chemical mechanism of ATP hydrolysis. In order to understand this mechanism, the ATPase domain of human TAP1 (NBD1) was expressed and purified, crystallized in nucleotide-free and transition-state complex forms and X-ray crystallographic studies were performed. The NBD1 protein was crystallized (i) in the nucleotide-free apo form; (ii) in complex with ADP-Mg(2+), mimicking the product-bound state; (iii) in complex with vanadate-ADP-Mg(2+), mimicking the ATP-bound state; and (iv) in complex with azide-ADP-Mg(2+), also mimicking the ATP-bound state. X-ray diffraction data sets were collected for apo and complexed NBD1 using an in-house X-ray diffraction facility at a wavelength of 1.5418 Å. The apo and complexed NBD1 crystals belonged to the primitive hexagonal space group P6(2), with one monomer in the asymmetric unit. Here, the crystallization, data collection and preliminary crystallographic analysis of apo and complexed NBD1 are reported.

Rv0864 (MoaC2) from Mycobacterium tuberculosis is one of the enzymes in the molybdenum cofactor (Moco) biosynthesis pathway. Together with MoaA, MoaC is involved in the conversion of guanosine triphosphate (GTP) to precursor Z, the first step in Moco synthesis. Full-length MoaC2 (17.5 kDa, 167 residues) was cloned in Escherichia coli and purified to homogeneity. Crystals of recombinant M. tuberculosis MoaC2 were grown by vapour diffusion using a hanging-drop setup. Diffracting crystals grew in a condition in which 3 µl protein solution at 10.5 mg ml(-1) was mixed with 1.5 µl reservoir solution (0.025 M potassium sodium tartrate tetrahydrate pH 8.0) and equilibrated against 1000 µl reservoir solution. Diffraction data extending to 2.5 Å resolution were collected at 100 K. The crystal belonged to the cubic space group P2(1)3, with unit-cell parameter 94.5 Å. Matthews coefficient (V(M)) calculations suggested the presence of two molecules in the asymmetric unit, corresponding to a solvent content of about 39%. Molecular-replacement calculations using the E. coli homologue as the search model gave an unambiguous solution.

Escherichia coli O157 paaR2-paaA2-parE2 constitutes a unique three-component toxin-antitoxin (TA) module encoding a toxin (ParE2) related to the classic parDE family but with an unrelated antitoxin called PaaA2. The complex between PaaA2 and ParE2 was purified and characterized by analytical gel filtration, dynamic light scattering and small-angle X-ray scattering. It consists of a particle with a radius of gyration of 3.95 nm and is likely to form a heterododecamer. Crystals of the ParE2-PaaA2 complex diffract to 3.8 Å resolution and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 142.9, c = 87.5 Å. The asymmetric unit is consistent with a particle of around 125 kDa, which is compatible with the solution data. Therefore, the ParE2-PaaA2 complex is the largest toxin-antitoxin complex identified to date and its quaternary arrangement is likely to be of biological significance.

Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism. In Plasmodium falciparum, three AK isoforms, namely PfAK1, PfAK2 and GTP:AMP phosphotransferase (PfGAK), have been identified. While PfAK1 and PfAK2 catalyse the conversion of ATP and AMP to two molecules of ADP, PfGAK exhibits a substrate preference for GTP and AMP and does not accept ATP as a substrate. PfGAK was cloned and expressed in Escherichia coli and purified using two-step chromatography. Brown hexagonal crystals of PfGAK were obtained and a preliminary diffraction analysis was performed. X-ray diffraction data for a single PfGAK crystal were processed to 2.9 Å resolution in space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 123.49, c = 180.82 Å, α = β = 90, γ = 120°.