Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

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 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.     

Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide.

Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.     

Crystallization and preliminary X-ray characterization of a soybean seed lipoxygenase

An isoenzyme of soybean (Glycine max L. Merrill cv. Provar) lipoxygenase (EC 1.13.11.12) has been crystallized using the vapor diffusion method. Crystals were grown from solutions of the protein (7 mg/ml) using 10 to 20% (w/v) polyethylene glycol 8000 in citrate/phosphate buffer (pH 5.7) containing 0.5% (w/v) n-octyl-beta-D-glucopyranoside. The crystals reached maximum dimensions of 0.3 mm x 0.2 mm x greater than 2 mm. The enzyme crystallized in space group C222(1) with unit cell dimensions a = 246 A, b = 193 A and c = 75 A. A calculated Vm value of 2.35 A3/dalton was obtained assuming two molecules per asymmetric unit. The density of the crystals was found to be 1.16 g/ml, which confirmed the presence of two molecules per asymmetric unit and indicated a solvent content of 47.5%.     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Crystallization and preliminary crystallographic data of a truncated derivative from the human c-Ha-ras protein

A truncated derivative of the human c-Ha-ras protein has been crystallized from polyethylene glycol 6000 solution by a vapour diffusion technique. The rectangular prism diffracts X-rays to at least 2.5 A resolution (1 A = 0.1 nm). The unit cell is monoclinic, space group P21, with unit cell parameters of a = 50.2 A, b = 110.9 A, c = 36.4 A, beta = 97.2 degrees. The unit cell contains four molecules.     

Crystallization and preliminary X-ray analysis of two different forms of mitochondrial creatine kinase from chicken cardiac muscle

Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.     

Preliminary X-ray analysis of crystals of plasminogen activator inhibitor-1

Crystals of bacterially expressed plasminogen activator inhibitor (PAI-1) suitable for X-ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 A, b = 47.82 A, c = 62.89 A, alpha = 90.00 degrees, beta = 106.90 degrees, gamma = 106.84 degrees. The diffraction limit is 2.3 A, and the unit cell contains two molecules of PAI-1. The crystals contain latent PAI-1 which can be partly reactivated by exposure to denaturants.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

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.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Purification, crystallization, and preliminary X-ray crystallographic analysis of thermus thermophilus V(1)-ATPase B subunit.

The gene of V(1)-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 A on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 A, b = 129.6 A, c = 92.7 A, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V(M) value is calculated as 2.8 or 2.1 A (3)/Da, respectively.     

Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.     

Crystallization and preliminary X-ray diffraction studies of bovine recombinant immune interferon

Reproducible conditions have been established for the crystallization of recombinant bovine immune interferon. Two cystalline forms of this protein were obtained. A tetragonal form, space group P422, with unit cell dimensions a = b = 59.0 A and c = 125.7 A and an orthorhombic form, space group P2(1)2(1)2(1), with unit cell dimensions a = 42.80 A, b = 79.90 A and c = 85.64 A were obtained under similar crystallization conditions. The orthorhombic form diffracts to 2.6 A resolution, contains a single interferon dimer in the asymmetric unit of structure and is suitable for X-ray diffraction analysis.     

Crystallization and preliminary X-ray study of horse pancreatic lipase

Horse (Equus caballus) pancreatic lipase (EC 3.1.1.3) has been crystallized using the hanging drop method of vapour diffusion at 20 degrees C. The best crystals were grown from an 8 mg/ml solution in 10 to 20% (w/v) polyethylene glycol 8000, 10 mM-MgCl2, 0.1 M-NaCl, 0.1 M-Mes buffer (pH 5.6). They reach dimensions of 0.8 mm x 0.4 mm x 0.6 mm. X-ray examination of the lipase crystals shows that they are orthorombic with a space group P2(1)2(1)2(1). Their cell dimensions are a = 79.8 A, b = 97.2 A c = 145.3 A. Two molecules per asymmetric unit give a Vm value of 2.82 A3/dalton (56% water content). Lipase crystals strongly diffract to at least 1.8 A resolution. Some molecular properties of horse lipase compared to those of the better-known porcine enzyme are also presented.     

Crystallization and preliminary characterization of CheY, a chemotaxis control protein from Escherichia coli

Single crystals of the 14.1-kDa cheY gene product from Escherichia coli have been grown from buffered ammonium sulfate solutions using the combined methods of microdialysis and pulsed diffusion. The crystals are of the monoclinic space group P2(l), have cell constants of a = 51.4 A, b = 112 A, c = 51.2 A, and beta = 107.3 degrees, and contain four molecules per asymmetric unit. They are stable to x-ray radiation and diffract beyond 3.0 A resolution.     

Preliminary X-ray analysis of crystals of murine adenosine deaminase

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.