The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.

The activity of interferon-γ (IFN-γ) relies on signal transduction, which is triggered by combination with the receptors interferon-γ receptor α chain (IFNGR1) and β chain (IFNGR2). Native recombinant chicken IFNGR1 (chIFNGR1; residues 25-237) was overexpressed in Escherichia coli, purified by refolding and crystallized using the vapour-diffusion technique. The crystals belonged to space group P6(5)22, with unit-cell parameters a = b = 64.1, c = 216.3 Å, α = β = 90, γ = 120°. The Matthews coefficient and solvent content were calculated as 2.67 Å(3) Da(-1) and 53.97%, respectively. X-ray diffraction data for chIFNGR1 were collected to 2.0 Å resolution at a synchrotron source.

The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion-tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni(2+)-NTA and Q-Sepharose ion-exchange columns and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from PA3346RS crystals to 2.0 Å resolution. The crystal belonged to space group P4(1) or P4(3), with unit-cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.

Phospholipases A(2) (PLA(2)s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA(2)s. BmooPLA(2)-I is an acidic, nontoxic and catalytic PLA(2) isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA(2)-I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C222(1), with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA(2)-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA(2) from B. jararacussu (BthA-I) and other toxic and nontoxic PLA(2)s may provide important insights into the functional aspects of this class of proteins.

The p38 MAP kinase pathway is an essential component of numerous cellular signalling networks which are usually activated in response to extracellular environmental stress conditions. In addition to the canonical activation, several alternative activation pathways have been identified for p38; one of these, in which p38 is initially phosphorylated on Tyr323 and consequently autoactivated, is exclusive to T cells and is induced by TCR activation. Intrinsically active and inactive mutants at position 323 have been developed in order to evaluate the structural changes that occur upon TCR-induced activation. In order to promote crystal growth, cross streak-seeding techniques were utilized. This technique has gained popularity in promoting crystal growth when spontaneous nucleation induces critical defects or is being entirely hindered. The crystal characteristics of some mutants were highly similar to those of the wild-type source seeds (form A). In contrast, other mutants crystallized spontaneously with a different space group and molecular packing (form B). One of the active mutants (Y323T) crystallized in both crystal forms, displaying different packing characteristics and significant differences in molecular conformation that were clearly dictated by the source seeds. This implies that the source seeds used in cross streak-seeding could, in some cases, impose bias on the structural outcome of the studied molecule. Such incidents could occur when the conformational freedom permits crystal packing while not reflecting the authentic structure.

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to2.5 Å resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Phosphoglycerate kinase (PGK) from methicillin-resistant Staphylococcus aureus MRSA252 has been cloned in pQE30 expression vector, overexpressed in Escherichia coli SG13009 (pREP4) cells and purified to homogeneity. The protein was crystallized from 0.15 M CaCl(2), 0.1 M HEPES-NaOH pH 6.8, 20%(w/v) polyethylene glycol 2000 at 298 K by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 45.14, b = 74.75, c = 58.67 Å, β = 95.72°. X-ray diffraction data have been collected and processed to a maximum resolution of 2.3 Å. The presence of one molecule in the asymmetric unit gives a Matthews coefficient (V(M)) of 2.26 Å(3) Da(-1) with a solvent content of 46%. The structure has been solved by molecular replacement and structure refinement is now in progress.

The overarching goal of this research project is to determine, for a subset of proteins, exact hydrogen positions using neutron diffraction, thereby improving H-atom placement in proteins so that they may be better used in various computational methods that are critically dependent upon said placement. In order to be considered applicable for neutron diffraction studies, the protein of choice must be amenable to ultrahigh-resolution X-ray crystallography, be able to form large crystals (1 mm(3) or greater) and have a modestly sized unit cell (no dimension longer than 100 Å). As such, γ-chymotrypsin is a perfect candidate for neutron diffraction. To understand and probe the role of specific active-site residues and hydrogen-bonding patterns in γ-chymotrypsin, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection. Time-of-flight neutron diffraction data were collected to 2.0 Å resolution at the PCS with ~85% completeness. Here, the first time-of-flight neutron data collection from γ-chymotrypsin is reported.

Metal ions often stabilize intermolecular contacts between macromolecules, thereby promoting crystallization. When interpreting a medium-resolution electron-density map of the catalytic domain of human sentrin-specific protease 1 (SENP1), a strong feature indicative of an ordered divalent cation was noted. This was assigned as Co(2+), an essential component of the crystallization mixture. The ion displays tetrahedral coordination by Glu430 and His640 from one molecule and the corresponding residues from a symmetry-related molecule. Analysis of the data derived from a previous structure of SENP1 suggested that Co(2+) had been overlooked and re-refinement supported this conclusion. High-throughput automated re-refinement protocols also failed to mark the Co(2+) position, supporting the requirement for the incorporation of as much information as possible to enhance the value of such protocols.