Preliminary crystallographic study of bluetongue virus capsid protein, VP7.

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.     

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

Trigonal crystals of porin from Escherichia coli

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.     

Crystallization and preliminary diffraction analysis of cholesterol esterase from Candida cylindracea

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 x 0.15 x 0.10 mm in size, diffract rotating anode generated x-rays to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P2(1) was found to have cell dimensions of a = 122.9 A, b = 101.0 A, c = 95.2 A and beta = 108.3 degrees. The asymmetric unit of the cell contains two dimers of 129 kDa each. A second crystal form, in the triclinic space group P1, has cell dimensions of a = 58.6 A, b = 88.7 A, c = 58.6 A, alpha = 93.3 degrees, beta = 113.8 degrees and gamma = 96.0 degrees, and has one dimer per asymmetric unit.     

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex

Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site. The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees. They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Crystallization of recombinant chitobiase from Serratia marcescens.

We are currently investigating the biochemical and structural properties of both chitin degrading enzymes chitinase and chitobiase from Serratia marcescens. Previously we have reported the first crystallization and characterization of chitinase crystals (Vorgias et al., 1992). In this communication we present the first crystallization of chitobiase. The protein was synthesized in Escherichia coli and purified to homogeneity using cation exchange chromatography and fast protein liquid chromatography. The crystals have the shape of small prisms and the space group is P2(1) with beta = 101.0 degrees and unit cell dimensions a = 63.2 A, b = 133.2 A, c = 55.1 A. They diffract X-rays to about 2.5 A resolution and are suitable for three-dimensional structural analysis.     

Crystallization of the met repressor from Escherichia coli

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).     

Crystallization and preliminary crystallographic study of DNA polymerase from Pyrococcus furiosus

A new member of archaeal DNA polymerase from Pyrococcus furiosus was crystallized. Diffraction data to 3.1 A of the selenomethionine-derivatized crystal were collected, and preliminary crystallographic study has been completed. The crystal belongs to the space group C2 with unit cell parameters of a = 93.2 A, b = 124.9 A, c = 87.7 A, alpha = 90 degrees , beta = 109.7 degrees , and gamma = 90 degrees . Assuming the presence of one molecule in the asymmetric unit, the solvent content of the crystal is estimated to be 54%, corresponding to a Matthews coefficient V(M) of 2.7A (3) Da(-1).     

Crystal structure of an anti-Lewis alpha Fab determined by molecular replacement methods

The anti-Lewis alpha mouse immunoglobulin CF4C4 (IgGl, k) Fab has been crystallized from 58% saturated ammonium sulfate in space group Pl; unit cell dimensions a = 43.4 A b = 41.7 A, c = 62.0 A, a = 72.7 degrees, beta = 96.6 degrees, gamma = 100.1 degrees. X-ray diffraction data have been measured beyond 3.0 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using as search models the constant and variable domains of the mouse immunoglobulin McPC603 (IgA, kappa) Fab. The crystallographic residual for the data 5.0 to 4.0 A, is 0.47. The approximate 2-fold axis relating the VL and the VH domains forms an angle of 164 degrees with the 2-fold axis relating the constant domains. The crystal packing is reasonable.     

Crystallization and preliminary x-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.     

Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% beta-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A, c = 78.7 A (hexagonal indexing) containing a dimer of Mr 112,000 in the asymmetric unit (Vm = 2.64 A3/dalton). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2(1). The first form has a = 88.1 A, b = 99.3 A, c = 114.6 A, beta = 119 degrees, the second form has a = 92.5 A, b = 117.6 A, c = 108.3 A, beta = 93.64 degrees. Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of Mr 224,000 in the asymmetric unit and have Vm values of 2.28 and 2.65 A3/dalton, respectively. All forms diffract to better than 3.0 A resolution while the second monoclinic form diffracts to approximately 1.8 A.     

Preliminary crystallographic study of an L-asparaginase from Vibrio succinogenes

Crystals of an L-asparaginase from Vibrio succinogenes were obtained with the hanging drop method from ammonium sulphate-containing solutions. The crystals belong to the orthorhombic space group P22(1)2(1) with unit cell dimensions of a = 71.3 A, b = 85.8 A, c = 114.0 A, and contain two tetrameric enzyme molecules per unit cell. There are two subunits in the asymmetric unit; a molecular dyad is coincident with the crystallographic dyad. The crystal lattice is similar to that reported for an Escherichia coli asparaginase. Rotation function calculations have revealed that the V. succinogenes enzyme has 222 point group symmetry in the crystal. The second and third molecular dyads differ, however, from the corresponding E. coli asparaginase dyads by approximately 40 degrees. The crystals diffract to at least 2.2 A resolution and are suitable for X-ray crystallographic structure determination.     

Preliminary crystallographic data on monomeric and dimeric hemoglobins from the sea cucumber, Molpadia arenicola.

Large single crystals of two distinct globin chains from coelomic cells of the sea cucumber Molpadia arenicola have been prepared and examined by x-ray crystallography. These hemoglobins exhibit a variety of ligand-dependent association states with the met-hemoglobins existing as monomers and liganded hemoglobins as dimers at physiological concentrations. Monomeric methemoglobin C chain crystallizes in space group P21, with a = 46.0 A, b = 45.3 A, c = 40.9 A, beta = 104.5 degrees, and one monomer per asymmetric unit. These crystals exhibit unusual spectroscopic behavior when examined with a polarizer, turning colorless in certain orientations. This implies that all the heme rings are nearly parallel within the crystals. Dimeric cyanmethemoglobin D chain crystallizes in space group P41212 (P43212), with a = b = 77.0 A, c = 61.5 A, and one-half a dimer per asymmetric unit. These homodimers thus possess a molecular 2-fold which is aligned with the crystallographic dyad.     

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5. The crystals belong to the monoclinic space group P2(1), with unit cell constant: a = 55.3 A, b = 59.4 A, c = 42.5 A, beta = 99.0 degrees; one complex moiety is present per asymmetric unit. The crystals diffract to 2.0 A resolution and are suitable for detailed X-ray crystallographic investigations.     

Preliminary x-ray diffraction characterization of crystalline rat liver dihydropteridine reductase

The pyridine dinucleotide-dependent dihydropteridine reductase from rat liver has been crystallized from various inorganic salts and from polyethylene glycol/ethanol mixtures using a vapor diffusion method. The crystal form most amenable to x-ray diffraction studies is monoclinic, space group C2, a = 224.3 A, b = 46.5 A, c = 94.9 A, beta = 99.0 degrees with two molecules in the crystallographic asymmetric unit. These crystals grow to over 1 mm in length, diffract to at least 2.3 A resolution, and are relatively stable to irradiation with x-rays.