Crystallization and preliminary X-ray studies of human recombinant interleukin-2

Two different forms of crystals (potentially) suitable for x-ray structure analysis were obtained for recombinant human interleukin-2 (IL-2) using ammonium sulfate as a precipitant in the pH range of 6.3-7.3 (in the case of hexagonal bipyramidal crystals) and 4.5-5.5 (in the case of plate crystals). The hexagonal bipyramidal crystal belongs to a hexagonal space group P6(2)22 or P6(4)22 with a = b = 105.8 A and c = 122.2 A. The crystal diffracts up to 3.4 A resolution and contains 2 or 3 IL-2 molecules in an asymmetric unit. The plate crystal belongs to an orthorhombic space group P2(1)2(1)2 with a = 47.9 A, b = 79.6 A, and c = 31.9 A. The crystal diffracts up to 2.5 A resolution and contains only 1 IL-2 molecule in an asymmetric unit. These facts reconfirmed crystallographically the high homogeneity of the present preparation of human recombinant IL-2.     

First crystallization of a phosphoenolpyruvate carboxylase from Escherichia coli

Two different forms of crystal for a phosphoenolpyruvate carboxylase from Escherichia coli were obtained by the hanging-drop vapor diffusion technique, using polyethylene glycol 4000 as precipitant. The hexagonal crystal in space group P6(2)22 (or P6(4)22) has cell dimensions of a = 131 A and c = 325 A, whereas the orthorhombic crystal in space group I222 has a = 119 A, b = 252 A and c = 83 A. A tetrameric molecule (396,244 Mr), a subunit of which contains 883 amino residues, has a crystallographic 2 symmetry in the hexagonal crystal or 222 symmetry in the orthorhombic crystal, respectively.     

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.     

Crystallization studies of cAMP-dependent protein kinase. Cocrystals of the catalytic subunit with a 20 amino acid residue peptide inhibitor and MgATP diffract to 3.0 A resolution.

Crystallographic studies of the catalytic subunit of cAMP-dependent protein kinase demonstrate that the presence of a 20 amino acid residue peptide inhibitor and MgATP during crystallization yields crystals with a different space group and, more significantly, makes an important difference in the quality of the resulting crystals. Under identical experimental conditions, the kinase crystallizes in a cubic space group P4(1)32 (a = b = c = 169.24 A), when no substrates or inhibitors are present, and in the hexagonal space group P6(1)22 (or P6(5)22) (a = b = 80.16 A, c = 288.07 A, alpha = beta = 90 degrees, gamma = 120 degrees) when a 20-amino acid residue peptide inhibitor and MgATP are present. Moreover, the hexagonal crystal diffracts to a resolution of 3.0 A, while the cubic crystals diffract to a resolution of 4.0 A.     

Preliminary crystallographic study of the Erythrina rubrinervia lectin

The galactose-specific lectin from Erythrina rubrinervia crystallizes in the hexagonal space group P6, (or P6(5)) with unit cell dimensions a = b = d = 135.1 A, c = 83.0 A. These parameters are compatible with the presence of a dimer with Mr = 60,000 in the asymmetric unit. The crystals are suitable for high-resolution X-ray analysis.     

Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus

Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6(1)22 (P6(5)22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 A resolution.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

Crystallization and preliminary x-ray investigation of purine-nucleoside phosphorylase from Escherichia coli

Crystals of purine-nucleoside phosphorylase from Escherichia coli have been grown from solutions of ammonium sulfate. The crystals are hexagonal with space group P6(1)22 or P6(5)22; the axes are alpha = 106.5 A and c = 241.3 A. The crystals are moderately stable to x-rays and diffract beyond 3.0-A resolution. It appears that the molecule, which is a hexamer, utilizes the 2-fold symmetry of the space group, resulting in three subunits/asymmetric unit.     

Crystallization of a photosensitive nitrile hydratase from Rhodococcus sp. N-771.

A photosensitive nitrile hydratase from Rhodococcus sp. N-771 has been crystallized in two different crystal forms in its inactive form. One crystal form belongs to an orthorhombic space group P2(1)2(1)2 with unit cell dimensions of a = 117.4 A, b = 145.7 A and c = 52.1 A, and the other form belongs to a hexagonal space group P6(3)22 with unit cell dimensions of a = 110.2 A and c = 412.1 A.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Preparation and X-ray characterization of four new crystal forms of jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.     

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.     

Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus.

Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.     

Crystals of Anabaena PCC 7119 ferredoxin-NADP+ reductase

Crystals of ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119 are grown in the presence of polyethylene glycol 6000 and beta-octyl glucoside. They belong to the hexagonal system. The cell parameters are a = b = 87.8 A, c = 92.7 A, space group P6(1) or P6(5), and a Vm of 3.0 A3/dalton for one molecule of 36,000 daltons per asymmetric unit. These crystals diffract strongly up to 1.9 A and are suitable for X-ray structural studies.     

Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme.

The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.     

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.     

New crystal form of recombinant murine interferon-beta

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.     

X-ray diffraction by crystals of beef heart ubiquinol: cytochrome c oxidoreductase.

Beef heart mitochondrial ubiquinol:cytochrome c oxidoreductase has been crystallized in the shape of hexagonal bipyramids. At present the crystals diffract X-rays to 4.7 A. From preliminary analysis the diffraction pattern appears to be consistent with space group P6(1)22 or P6(5)22 and with unit cell parameters a = b = 212 A and c = 352 A.     

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.     

Crystallization and preliminary X-ray crystallographic analysis of the pediocin immunity protein (PedB) from Pediococcus pentosaceus at 1.35 A resolution

PedB, a bacterial immunity protein conferring immunity to a newly identified pediocin (pediocin PP-1), was crystallized by the hanging-drop vapor diffusion method at 296 K. A 1.35 A data set has been collected from a single crystal at 100 K using synchrotron-radiation source. The PedB crystals belong to the hexagonal space group P6(2) or P6(4), with unit cell parameters a = b = 62.2, c = 39.9 A. Analysis of the packing density shows that the asymmetric unit probably contains one molecule with a solvent content of 33.8%.