Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 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.     

Purification,crystallization, and preliminary X-ray analysis of Bacillus subtilis ferrochelatase

Bacillus subtilis ferrochelatase (EC 4.99.1.1), the final enzyme in protoheme IX biosynthesis, was produced with an inducible T7 RNA polymerase expression system in Escherichia coli and purified from the soluble cell fraction. It was crystallized from polyethylene glycol solution using the microseeding technique. The crystals diffract to a minimum Bragg spacing of 2.1 Å. The space group is P4₂ with unit cell dimensions a = b = 50.2 Å, c = 120.1 Å. © 1995 Wiley-Liss, Inc.     

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,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 diffraction analyses of the bacterial chemotaxis receptor modifying enzymes

Bacterial chemotaxis receptor modifying enzymes from Salmonella typhimurium have been crystallized using microseeding techniques. The crystals of the S-adenosyl-L -methionine-dependent methyl transferase, CheR, belong to the monoclinic space group P2₁ with cell constants a = 55.1 Å, b = 48.1 Å, c = 63.1 Å, β = 112.3°. The crystals of the catalytic domain of the methylesterase, CheB, belong to the trigonal space group P3₂21 or P3₁21 with unit cell dimensions of a = b = 63.4 Å, c = 86.8 Å. Both crystals contain one molecule per asymmetric unit and have calculated Matthews' volumes of 2.4 ų/Da. © 1995 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies of a bifunctional 5,10-methenyl/methylene tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 ų/Da. The crystals diffract to at least 2.8 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12-C at the NSLS. © 1997 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.     

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.     

Molecular Symmetry of the ClpP Component of the ATP-dependent Clp Protease,anEscherichia coliHomolog of 20 S Proteasome

The ClpP component Clp protease fromEscherichia colihas been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystal belongs to the monoclinic space groupP2₁with unit cell dimensions ofa=196.9 Å,b=104.3 Å,c=162.4 Å and β=98.3°. The X-ray diffraction pattern extends at least to 2.5 Å Bragg spacing when exposed to CuKα X-rays. Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry. This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)₁₄, consisting of two heptamers, (ClpP)₇stacked on top of each other in a head-to-head fashion. The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (V_M) of 2.73 ų/Da and a solvent content of 54.9% (v/v). Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit. The Patterson function suggests that a translation ofx=0.5 andy=0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit. And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18° around the 7-fold axis.     

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 crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl^? buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset. © 1995 Wiley-Liss, Inc.     

Low resolution structure of adenylate kinase

A low resolution model of adenylate kinase has been derived from a 6 Å electron density map. The molecular shape can be described approximately as an oblate ellipsoid with dimensions 40 Å × 40 Å × 30 Å. The molecule is composed of two globular units separated by a 10 Å deep cleft. In contrast to the bigger unit, the smaller globule appears to contain a high amount of α-helical structure. The location of the active centre is discussed.The crystals used for X-ray diffraction analysis belong to one of the enantiomorphic trigonal space groups P3₁21 or P3₂21, with one molecule in the asymmetric unit. The phase determination was based on four isomorphous heavy atom derivatives. Frequent transitions between different crystal forms complicate the analysis.     

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 X-ray studies of ornithine decarboxylase from Trypanosoma brucei

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2₁ and have cell constants of a = 66.3 Å, b = 151.8 Å, c = 83.7 Å, and β = 101.2°. While larger crystals are twinned, smaller crystals (0.4 × 0.3 × 0.05 mm³) are single.     

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 × 0.25 × 1.20 mm. The space group of the crystal is I4₁22, with unit cell dimensions a = b = 56.88 Å, c = 230.11 Å, α = β = γ = 90°, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/σ(I) ratio for data at 3.0 Å resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2; the unit cell axes are a = 36.5 Å and c = 221.9 Å. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic data for class I deoxyribose-5-phosphate aldolase from Escherichia coli: An Application of Reverse Screening

X-ray quality crystals of class I deoxyribose-5-phosphate aldolase from Escherichia coli have been obtained for the unliganded enzyme and in complex with its substrate, 2-deoxyribose-5-phosphate. The enzyme catalyzes the reversible cleavage of 2-deoxyribose-5-phosphate to acetaldehyde and D-glyceraldehyde-3-phosphate. The unliganded and complex crystals are prismatic long rods and belong to the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 183.1 Å, b = 61.4 Å, c = 49.3 Å and a = 179.2 Å, b = 60.5, Å, c = 49.1 Å, respectively. Two molecules in the asymmetric unit are related by a noncrystallo-graphic 2-fold axis. The crystals are stable in the X-ray beam and diffract to at least 2.6 Å. A new method, reverse screening, designed to minimize protein utilization during the screening process was used to determine supersaturation and crystallization conditions. © 1995 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.