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The purification, crystallization and preliminary X-ray diffraction analysis of RNase HIII from S. aureus is presented. Crystals that diffracted to 2.6 Å resolution in space group P212121 were only obtained after removal of the hexahistidine tag.

As part of collaborative efforts to characterize virulence factors from Staphylococcus aureus, methods for the large-scale recombinant production of RNase HIII from S. aureus subspecies MRSA252 (Sa-RNase HIII) have been developed. RNase HIII-type ribonucleases are poorly characterized members of the RNase H group of endonucleases which hydrolyze RNA from RNA/DNA hybrids and are thought to be involved in DNA replication and repair. They are characterized by N-terminal extensions of unknown function that do not share sequence homology with the N-terminal extensions of bacterial RNases HI and RNases HII. Sa-RNase HIII was crystallized in the orthorhombic space group P212121, with unit-cell parameters a = 48.9, b = 74.2, c = 127.5 Å, and diffracted to 2.6 Å resolution.

Type 1 RNase H from the hyperthermophilic archaeon S. tokodaii 7 was overproduced in E. coli, purified, and crystallized. Preliminary crystallographic studies indicated that the crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å.

Crystallization and preliminary crystallographic studies of type 1 RNase H from the hyperthermophilic archaeon Sulfolobus tokodaii 7 were performed. A crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.5 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to space group P43, with unit-cell parameters a = b = 39.21, c = 91.15 Å. Assuming the presence of one molecule in the asymmetric unit, the Matthews coefficient V M was calculated to be 2.1 Å3 Da−1 and the solvent content was 40.5%. The structure of a selenomethionine Sto-RNase HI mutant obtained using a MAD data set is currently being analysed.

This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6222 and diffracted to a resolution of 2.4 Å.

Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6222, with unit-cell parameters a = b = 145.97, c = 118.03 Å.

A thermostable multicopper oxidase from Thermus thermophilus HB27 (Tth-MCO) has been successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods.

A thermostable multicopper oxidase from Thermus thermophilus HB27 (Tth-MCO) was successfully crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. Crystallization conditions and preliminary X-ray diffraction data to 1.5 Å resolution obtained using synchrotron radiation at 100 K are reported. The crystals belonged to space group C2221, with unit-cell parameters a = 93.6, b = 110.3, c = 96.3 Å. A monomer in the asymmetric unit yielded a Matthews coefficient (V M) of 2.60 Å3 Da−1 and a solvent content of 53%. An inactive enzyme form, apo-Tth-MCO, was also crystallized and diffraction data were collected to 1.7 Å resolution. In addition, a second inactive form of the enzyme, Hg-Tth-MCO, was obtained by soaking apo-Tth-MCO crystals with mercury(II) chloride and data were collected to a resolution of 1.7 Å.

Purification and preliminary X-ray crystallographic analysis of maleylacetate reductase encoded by the pnpD gene is reported.

Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P21212, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 Å. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 Å resolution, respectively.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed.

Glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis was crystallized and preliminary crystallographic studies of the crystals were performed. Crystals were grown at 293 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.4 Å resolution using synchrotron radiation at station BL44XU of SPring-8. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 217.48, c = 66.48 Å. Assuming the presence of two molecules in the asymmetric unit, the V M value was 2.7 Å3 Da−1 and the solvent content was 54.1%. The protein was also cocrystallized with substrates and diffraction data were collected to 2.7 Å resolution.

BTL2, a thermostable enantioselective biocatalyst of interest for industrial applications, has been crystallized using the sitting-drop vapour-diffusion method. Preliminary X-ray data to 2.2 Å resolution allowed the determination of its unit-cell parameters and space group and initiation of its structure determination.

Bacillus thermocatenulatus lipase 2 (BTL2) is a thermoalkalophilic lipase that has been reported as an enantioselective biocatalyst for diverse reactions and that heads a group of enzymes that share high resistance towards many inactivation agents (heat, organic solvents, pH etc.). This makes BTL2 an important research target because of its potential industrial applications. BTL2 was cloned and overexpressed in Escherichia coli, purified and concentrated for crystallization using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 13% MPD and 0.2 M ammonium acetate in 0.05 M sodium citrate pH 5.5–5.6. The crystals, which belonged to the orthorhombic space group I222 with unit-cell parameters a = 73.07, b = 129.08, c = 127.49 Å, allowed the collection of an X-ray data set to 2.2 Å resolution.

The C-terminal RNase III domain (RNase IIIb) of human Dicer has been expressed, purified and crystallized by the sitting-drop vapour-diffusion method.

Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2221, with unit-cell parameters a = 88.6, b = 199.7, c = 119.6 Å, and diffracted X-rays to 2.0 Å resolution. The asymmetric unit contained three molecules of the RNase IIIb and the solvent content was 67%.

A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized in two forms using the sitting-drop vapour-diffusion method. Both crystals diffracted to approximately 1.3 Å resolution.

A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 Å, α = 71.2, β = 81.0, γ = 64.0°, and diffracted to 1.3 Å resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 Å, and diffracted to 1.3 Å resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon T. kodakaraensis were performed.

Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.

The cloning, expression, purification, crystallization and preliminary X-ray characterization of an l-N-carbamoylase from G. stearothermophilus are described.

N-Carbamoyl-l-amino-acid amidohydrolases (l-N-carbamoylases; EC 3.5.1.87) hydrolyze the carbon–nitrogen bond of the ureido group in N-carbamoyl-l-α-amino acids. These enzymes are commonly used in the production of optically pure natural and non-natural l-amino acids using the ‘hydantoinase process’. Recombinant l-N-carbamoylase from Geobacillus stearothermophilus CECT43 has been expressed, purified and crystallized by hanging-drop vapour diffusion. X-­ray data were collected to a resolution of 2.75 Å. The crystals belonged to space group P21212, with unit-cell parameters a = 103.2, b = 211.7, c = 43.1 Å and two subunits in the asymmetric unit.

The [NiFe]-hydrogenase maturation protein HypE was purified and crystallized. Crystals of HypE suitable for data collection diffracted to 1.55 Å resolution.

The hydrogenase maturation protein HypE is involved in the biosynthesis of the CN ligands of the active-site iron of [NiFe] hydrogenases using carbamoylphosphate as a substrate. Here, the crystallization and preliminary crystallographic analysis of HypE from Thermococcus kodakaraensis KOD1 are reported. Crystals of HypE (338 amino acids, 35.9 kDa) have been obtained by the sitting-drop vapour-diffusion method using 2-methyl-2,4-pentanediol (MPD) as a precipitant. The crystals belong to space group P21212, with unit-cell parameters a = 88.3, b = 45.8, c = 75.1 Å. There is one HypE molecule in the asymmetric unit. A complete native X-ray diffraction data set was collected to a maximum resolution of 1.55 Å at 100 K.

Preliminary X-ray crystallographic study of a proline-specific aminopepitdase from Aneurinibacillus sp, strain AM-1 was carried out.

To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination.

The expression, purification, crystallization and preliminary crystallographic analysis of recombinant β-mannanase from B. licheniformis strain DSM13 are described.

The mannan endo-1,4-β-mannosidase (ManB) from Bacillus licheniformis strain DSM13 was overexpressed in Escherichia coli. Purification of the thermostable and alkali-stable recombinant mannanase yielded approximately 50 mg enzyme per litre of culture. Crystals were grown by hanging-drop vapour diffusion using a precipitant solution consisting of 12%(w/v) PEG 8000, 0.2 M magnesium acetate tetrahydrate and 0.1 M MES pH 6.5. The protein crystallized in the monoclinic space group P21, with two molecules per asymmetric unit and unit-cell parameters a = 48.58, b = 91.75, c = 89.55 Å, β = 98.29°, and showed diffraction to 2.3 Å resolution.

The crystallization of an alginate-binding protein isolated from Sphingomonas sp. A1 and the results of preliminary crystallographic analysis of this protein are presented.

Sphingomonas sp. A1, a Gram-negative bacterium, directly internalizes alginate macromolecules through a mouth-like pit that is present on its cell surface. The alginate-binding protein Algp7, which was found to be localized on the cell surface, contributes to the accumulation of alginate in the pit. Algp7 was crystallized at 293 K by means of the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as a crystallizing agent. Preliminary X-ray analysis showed that the Algp7 crystal belonged to space group P212121, with unit-cell parameters a = 50.1, b = 98.0, c = 100.1 Å, and that it diffracted to 2.8 Å resolution.

NADH:rubredoxin oxidoreductase from C. acetobutylicum was expressed in E. coli, purified and crystallized. X-ray diffraction data were collected to a resolution of 2.1 Å.

NADH:rubredoxin oxidoreductase (NROR), an O2-inducible protein, is a versatile electron donor for scavengers of O2 and reactive oxygen species (ROS) in Clostridium acetobutylicum. Recombinant NROR was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the sitting-drop vapour-diffusion method at 293 K. Preliminary crystallo­graphic analysis revealed that the crystals belonged to space group P4122 or P4322, with unit-cell parameters a = b = 98.6, c = 88.3 Å, and diffracted to 2.1 Å resolution. Assuming that the crystals contained one molecule per asymmetric unit, the Matthews coefficient was calculated to be 2.7 Å3 Da−1 and the solvent content to be 54.1%.

Two enzymes responsible for arginine biosynthesis in M. tuberculosis were expressed in Escherichia coli, then purified to homogeneity. Preliminary X-ray analysis of diffraction-quality crystals grown from each enzyme are reported.

The gene products of two open reading frames from Mycobacterium tuberculosis (Mtb) have been crystallized using the sitting-drop vapour-diffusion method. Rv1652 encodes a putative N-acetyl-γ-glutamyl-phosphate reductase (MtbAGPR), while the Rv1656 gene product is annotated as ornithine carbamoyltransferase (MtbOTC). Both MtbAGPR and MtbOTC were expressed in Escherichia coli, purified to homogeneity and crystallized. Native data for each crystal were collected to resolutions of 2.15 and 2.80 Å, respectively. Preliminary X-ray data are presented for both enzymes.

UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K.

UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å3 Da−1 and a solvent content of 43%.

The bacterial PPM phosphatase RsbX from B. subtilis was expressed in E. coli, purified and crystallized. The crystal belonged to space group P1 and diffracted to 1.06 Å resolution.

RsbX from Bacillus subtilis is a manganese-dependent PPM phosphatase and negatively regulates the signal transduction of the general stress response by the dephosphorylation of RsbS and RsbR, which are activators of the alternative RNA polymerase σ factor SigB. In order to elucidate the structural–functional relationship of its Ser/Thr protein-phosphorylation mechanism, an X-ray crystallographic diffraction study of RsbX was performed. Recombinant RsbX was expressed in Escherichia coli, purified and crystallized. Crystals were obtained using the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 1.06 Å resolution with an R merge of 8.1%. The crystals belonged to the triclinic space group P1, with unit-cell parameters a = 33.3, b = 41.7, c = 68.6 Å, α = 98.8, β = 90.0, γ = 108.4°.

A soluble domain spanning amino-acid residues 16–197 of the Drosophila Dribble protein was overexpressed in E. coli, purified and crystallized. X-ray diffraction data were collected to beyond 2 Å resolution.

Dribble (DBE) is a Drosophila protein that is essential for ribosome biogenesis. Bioinformatics analysis revealed a folded central domain of DBE which is flanked by structural disorder in the N- and C-terminal regions. The protein fragment spanning amino-acid residues 16–197 (DBE16–197) was produced for structural determination. In this report, the crystallization and preliminary X-­ray diffraction data analysis of the DBE16–197 protein domain are described. Crystals of DBE16–197 were grown by the sitting-drop vapour-diffusion method at 289 K using ammonium phosphate as a precipitant. The crystals belonged to space group P212121. Data were collected that extended to beyond 2 Å resolution.

The crystallization of rice α-amylase/subtilisin bifunctional inhibitor is reported.

Rice bifunctional α-amylase/subtilisin inhibitor (RASI) can inhibit both α-­amylase from larvae of the red flour beetle (Tribolium castaneum) and subtilisin from Bacillus subtilis. The synthesis of RASI is up-regulated during the late milky stage in developing seeds. The 8.9 kDa molecular-weight RASI from rice has been crystallized using the hanging-drop vapour-diffusion method. According to 1.81 Å resolution X-ray diffraction data from rice RASI crystals, the crystal belongs to space group P21212, with unit-cell parameters a = 79.99, b = 62.95, c = 66.70 Å. Preliminary analysis indicates two RASI molecules in an asymmetric unit with a solvent content of 44%.

The crystallization and preliminary X-ray characterization of the polysaccharide lyase family 11 rhamnogalacturonan lyase are presented.

Rhamnogalacturonan lyases degrade rhamnogalacturonan I, a major component of pectin, through a β-elimination reaction. YesW from Bacillus subtilis strain 168 is a novel rhamnogalacturonan lyase classified into polysaccharide lyase family 11 (PL-11). The enzyme was crystallized at 293 K using the sitting-drop vapour-diffusion method with 2-methyl-2,4-pentanediol (MPD) as a precipitant. Preliminary X-ray analysis revealed that the YesW crystals belong to space group P21 and diffract to 2.40 Å resolution, with unit-cell parameters a = 56.7, b = 105.6, c = 101.4 Å, β = 94.9°. This is the first report on the crystallization and preliminary X-ray analysis of a family PL-11 rhamnogalacturonan lyase.

Human common-type acylphosphatase was crystallized in space group P212121, with unit-cell parameters a = 42.58, b = 47.23, c = 57.26 Å. A complete diffraction data set was collected to 1.45 Å.

Human acylphosphatase, an 11 kDa enzyme that catalyzes the hydrolysis of carboxyl phosphate bonds, has been studied extensively as a model system for amyloid-fibril formation. However, the structure is still not known of any isoform of human acylphosphatase. Here, the crystallization and preliminary X-­ray diffraction data analysis of human common-type acylphosphatase are reported. Crystals of human common-type acylphosphatase have been grown by the sitting-drop vapour-diffusion method at 289 K using polyethylene glycol 4000 as precipitant. Diffraction data were collected to 1.45 Å resolution at 100 K. The crystals belong to space group P212121, with unit-cell parameters a = 42.58, b = 47.23, c = 57.26 Å.

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the transcriptional repressor SirR (staphylococcal iron regulator) from M. tuberculosis are reported.

SirR, a metal-dependent transcriptional repressor from Mycobacterium tuber­culosis (Rv2788), was cloned in pQE30 expression vector with an N-­terminal His6 tag for heterologous overexpression in Escherichia coli M15 (pREP4) cells and purified to homogeneity using chromatographic procedures. The purified protein was crystallized using the sitting-drop vapour-diffusion technique. The crystals belonged to the tetragonal space group P41212/P43212, with unit-cell parameters a = 105.21, b = 105.21, c = 144.85 Å. The X-ray diffraction data were processed to a maximum resolution of 2.5 Å. The Matthews coefficient suggests the presence of two (V M = 4.01 Å3 Da−1) to four (V M = 2.0 Å3 Da−1) molecules in the asymmetric unit. Calculation of the self-rotation function shows a crystallographic fourfold symmetry axis along the z axis (χ = 90°) and also a twofold symmetry axis around the z axis (χ = 180°).

Preliminary crystallographic analysis of KaiC-like protein PH0186 from Pyrococcus horikoshii OT3 is reported.

KaiC is the central protein in the circadian-clock system of cyanobacteria. A selenomethionine-labelled KaiC-homologous protein from Pyrococcus horikoshii OT3 (PH0186; 28 kDa) was crystallized by the sitting-drop vapour-diffusion method using ethanol as a precipitant. The crystals diffracted X-rays to beyond 2.0 Å resolution using a synchrotron-radiation source. The space group of the crystals was determined to be C2, with unit-cell parameters a = 173.7, b = 51.8, c = 97.5 Å, β = 122.8°. The crystal contains three molecules in the asymmetric unit (V M = 2.2 Å3 Da−1) and has a solvent content of 43.5%. Sixfold noncrystallographic symmetry was identified from self-rotation calculations, assuming the presence of a hexamer in the crystal.