Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

Crystallization and preliminary x-ray studies of I-CreI: a group I intron-encoded endonuclease from C. reinhardtii

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19–24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 Å, c = 67.4 Å. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 ų/Da. The crystals diffract to at least 3.0 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss Inc.     

Preliminary X-ray crystallographic analysis of Tritrichomonas foetus inosine-5′-monophosphate dehydrogenase

Inosine-5′-monophosphate dehydrogenase (IMPDH) from the protozoan parasite Tritrichomonas foetus has been expressed in E. coli and crystallized. Crystals were grown to 0.1 mm in each dimension in 18 to 72 h using ammonium sulfate and low-molecular-weight polyethylene glycols. The crystals belong to the cubic space group P432 with unit cell edge = 157.25 Å. The enzyme is a homotetramer with each monomer having a molecular weight of 55,534 Da. There is one monomer per asymmetric unit, based on a volume/mass ratio of 2.7 ų/Da and self-rotation analysis. The crystals are adequately stable to allow a complete data set to be collected from a single crystal. Complete native data sets have been collected to 2.3 Å resolution at 4°C using synchrotron radiation. High-quality complete data extending to 3.0 Å resolution have been collected from crystals of four putative derivatives, and the data appear to be isomorphous with that of the native crystals in each case. Efforts to solve the derivatives for use in MIR phasing are underway. © 1995 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Characterization of crystals of a cytochrome oxidase (nitrite reductase) from Pseudomonas aeruginosa by x-ray diffraction and electron microscopy

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m-ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P2₁2₁2 with unit cell dimensions of 92 Å × 115 Å × 76 Å. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm³), coupled with the unit cell volume (804,000 ų), indicates that there is one subunit (~63,000 Mr) per asymmetric unit. X-ray diffraction data which were limited to 12 Å resolution due to small crystal size were obtained for the hk0 and 0kl zones using precession photography. Amplitude and phase data for the hk0, 0kl, and h0l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 Å resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 Å. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 Å) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.     

Crystallization and preliminary X-ray diffraction studies of a bacterial flavohemoglobin protein

A flavohemoglobin protein (FHP) was isolated from Alcaligenes eutrophus and has been crystallized by vapor diffusion methods using PEG 3350 as precipitant. The crystals of the FAD- and heme-containing protein belong to the monoclinic space group P2₁ with unit cell parameters of 52.2 Å, 85.8 Å, 103.9 Å, and 81.8° corresponding to two molecules per asymmetric unit. The crystals diffract at least to a resolution of 2.0 Å and are suitable for an X-ray structure analysis. © 1995 Wiley-Liss, Inc.     

Purification and crystallization of a schistosomal glutathione S-Transferase

The 26-kDa glutathione S-transferase from Schistosoma japonica (Sj26), a potential antischistosomal vaccine antigen, has been crystallized in an unligated form. Sj26 was recombinantly produced in E. coli without using a glutathione affinity column to facilitate preparation of unligated enzyme. The recombinant protein contains all 218 residues of Sj26^1,2 and an additional 13 residues linked to the C-terminus. Crystals of recombinant Sj26 were obtained by the vapor diffusion method using ammonium sulfate as the precipitant at pH 5.6. The crystals belong to the hexagonal space group P6₃22 with unit cell dimensions a = b = 125.2 Å and c = 72.0 Å and contain one Sj26 monomer per asymmetric unit. A complete native diffraction data set has been obtained to 2.4 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization,characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.16) was purified from the liver of Rana catesbeiana (bullfrog). Crystals of the protein have been obtained at 22°C by the hanging drop vapor diffusion technique, with polyethylene glycol as precipitant. Tetragonal crystals of about 0.3 × 0.3 × 0.7 mm diffract at room temperature to at least 3.5 Å using a conventional source and are stable to X-radiation for about 12 h. Therefore, these crystals are suitablefor high resolution studies. The space group is P4₁2₁2 (or its enantiomorph P4₃2₁2), with unit cell dimensions a = b = 291.6 Å and c = 189.4 Å. Density packing considerations areconsistent with the presence of 4-6 monomers (M_r of the monomer, 160,000) in the asymmetric unit. Amino-terminal sequence of the enzyme and of a chymotryptic fragment of 73.7 kDa containing the COOH-terminus has been obtained. The extensive sequence identity with rat and human carbamoyl phosphate synthetase I indicates the relevance for mammals of structural data obtained with the frog enzyme. © 1995 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies on the rhizome lectin from stinging nettle and its complex with NN′N″-triacetylchitotriose

Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P2₁2₁2₁ with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P2₁2₁2₁ with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic study of human CksHs1: A cell cycle regulatory protein

The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHsl crystals were grown in the presence of vanadate, a phos-phatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6₁22 with unit cell dimensions: a=b=94 Å, c=131.6 Å, and γ =120. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 Å. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual V_m value of 4.4 ų/Da or a solvent content of 72%. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary x-ray diffraction studies of a monoclonal antibody fab fragment against foot-and-mouth disease virus and of its complex with the main antigenic site peptide

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot-and-mouth disease virus (FMDV) C-SBcl and its complex with a peptide, corresponding to the major antigenic site of FMDV (VPl residues 136–150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 Å resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 Å, b = 89.12 Å, c = 64.04 Å, and β = 112.9° and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 Å resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 Å, b = 60.67 Å, c = 143.45 Å, and β = 95.4°, Density packing considerations indicate that there are two Fab molecules in the asymmetric unit. © 1994 John Wiley & Sons, Inc.     

Crystallization and preliminary X-ray diffraction study of the green flavoenzyme 5-Hydroxyvaleryl-CoA dehydratase/dehydrogenase from Clostridium aminovalericum

The bifunctional flavoenzyme 5-hydroxyvaleryl-CoA dehydratase/ dehydrogenase has been crystallized from solutions containing ammonium sulfate (form I) or polyethylene glycol (form II) as precipitant. In both cases, the crystals grew in the monoclinic space group C2. The unit cell dimensions for form I crystals were determined as a = 162.8 Å, b = 71.8 Å, c = 83.5 Å, β = 109.1°. Corresponding values for form II crystals were a = 161.2 Å, b = 71.6 Å, c = 82.2 Å, β = 109.3°. In both cases most probably there are two monomers per asymmetric unit. The crystals diffract to about 2 Å resolution and are rather stable in the X-ray beam. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1

A nonglycosylated (N₃₀QN₇₈Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2₁, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.     

Preliminary X-ray diffraction analysis of crystals of turnip yellow mosaic virus (TYMV)

Turnip yellow mosaic virus (TYMV) was purified from Chinese cabbage and crystallized in a form that permits high resolution structural analysis using X-ray diffraction. The crystals have a hexagonal bipyramidal morphology and often achieve dimensions of 1.0 × 1.0 × 0.5 mm. The crystals appear to be of hexagonal space group P6₂22 with a = b = 525 Å, c=315 Å, but we cannot strictly rule out the possibility that the space group is P622. They appear different than any crystals of TYMV previously reported. There are three T = 3 virus particles in the unit cell, which implies that one quarter of the particle, or 45 protein subunits, comprises the asymmetric unit of the crystal. Native data have been collected using synchrotron radiation to a resolution of 3.2 Å. © 1995 Wiley-Liss, Inc.     

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Cocrystallization of Lysyl–tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNA^lYS or Escherichia coli tRNA^lys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18–22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 Å, c = 119 Å) and diffract to at least 2.7 Å resolution. However the homocomplex is of space group P42₁2 and the heterocomplex of space group I422. © 1995 Wiley-Liss, Inc.     

Preliminary X-ray diffraction analysis of crystals of tomato aspermy virus (TAV)

Tomato aspermy virus (TAV) is a member of the T = 3 cucumovirus group, and the chrysanthemum strain (C-TAV) has been crystallized in a form suitable for X-ray structural analysis. The crystals, which grow in 14–17% ethanol at pH 8.5, are of orthorhombic space group I222 with unit cell dimensions of a = 295.1 Å, b = 320.5 Å, and c = 383.6 Å. There are two T = 3 virus particles in the unit cell, which means that they must be centered at 0,0,0 and 1/2, 1/2, 1/2 with icosahedral 222 symmetry elements coincident with crystallographic symmetry operators. The asymmetric unit of the crystals, therefore, contains one quarter of a virus particle, or 45 capsid subunits. Native diffraction data to 4 Å resolution have been collected using synchrotron radiation, though data appear to be present beyond that resolution. © 1995 Wiley-Liss, Inc.