Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2.

The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.     

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.     

Crystallographic studies of 3-ketoacylCoA thiolase from yeast Saccharomyces cerevisiae

Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained. The crystals diffract to at least 2.4 A. The space group of these crystals is P2(1)2(1)2(1), with cell dimensions a = 71.8 A, b = 93.8 A and c = 119.9 A. There is one dimer per asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Preliminary X-ray studies of the tetra-heme cytochrome c3 and the octa-heme cytochrome c3 from Desulfovibrio gigas

A tetra-heme and an octa-heme cytochrome c3 from the sulfate bacterium Desulfovibrio gigas have been crystallized. Diffraction quality crystals of the tetra-heme cytochrome are obtained from solution by the addition of polyethylene glycol at pH 6.5. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell parameters a = 42.27 A, b = 52.54 A and c = 52.83 A. The octa-heme cytochrome crystals develop from low ionic strength solutions of phosphate or Tris-Cl in the pH range 6.2-7.6. The crystals belong to the trigonal system, space group P3(1) or the enantiomorph P3(2), with unit cell parameters a = b = 57.4 A, c = 97.3 A, gamma = 120 degrees. Single crystal diffraction studies of the structures of these two low-potential cytochromes are in progress.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.     

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.     

Single crystals of V amylose complexed with isopropanol and acetone

Single crystals of amylose complexed with isopropanol or acetone were prepared by adding these precipitants to a metastable aqueous solution of amylose. With both precipitants, similar micrometre sized platelet crystals were obtained. They gave indistinguishable electron diffraction diagrams which could be indexed in an orthorhombic unit cell, with a = 28.26 A, b = 29.30 A, c = 8.01 A and in a space group P2(1)2(1)2(1) or P2(1)2(1)2. Within the unit cell, the amylose chains are organized in antiparallel pairs of parallel 6(5) amylose helices occupying 70% of the cell content, the remaining 30% consisting of isopropanol/acetone and water, with an estimate of 10 isopropanol/acetone molecules for 52 water molecules per unit cell. If the crystals are suspended in pure isopropanol at various temperatures or in pure methanol at room temperature, they undergo a de-solvation process that ultimately converts them into VH amylose. De-solvation with isopropanol left the crystals intact whereas with methanol, they became cracked during the shrinkage. An explanation is proposed for such difference.     

Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants

The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized. Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies. The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A. These crystals diffract to at least 1.8 A resolution. The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees. These crystals diffract to at least 2.0 A resolution.     

Characterization of bacterial photosynthetic reaction center crystals from Rhodopseudomonas sphaeroides R-26 by X-ray diffraction

An orthorhombic crystal form (P2(1)2(1)2(1)) of the reaction center from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 has been characterized. The crystals were grown from polyethylene glycol; the unit cell dimensions are a = 142.2 A, b = 139.6 A, and c = 78.7 A; and they contain one reaction center in each crystallographic asymmetric unit. The crystals diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Preliminary crystallographic study of the alpha-D-galactose-specific lectin from jack fruit (Artocarpus integra) seeds

Crystals of the alpha-D-galactose-specific lectin from Jack fruit (Artocarpus integra) have been obtained from polyethylene glycol 400 solutions. The crystals are orthorhombic, space group P2(1)2(1)2 with a = 77.09 A, b = 123.3 A and c = 78.73 A (1 A = 0.1 nm) and have one 39,500 Mr tetramer per asymmetric unit. The crystals diffract to at least 2.8 A on precession photographs.     

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.     

Crystallization of insecticyanin from the hemolymph of the tobacco hornworm Manduca sexta L. in a form suitable for a high resolution structure determination

Insecticyanin, a blue biliprotein from the tobacco hornworm Manduca sexta, has been crystallized in a form suitable for a high resolution x-ray analysis. The crystals grow by vapor diffusion against solutions of polyethylene glycol 8000 at pH 5.5. They belong to the space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 115.0 A; c = 71.1 A. Insecticyanin is believed to be a tetramer in solution; there are two subunits per asymmetric unit. The crystals diffract to at least 2.2 A resolution and appear reasonably resistant to radiation damage.     

Crystallization and preliminary X-ray diffraction data of two crystal forms of bovine heart creatine kinase

Two crystal forms of bovine heart creatine kinase, which are suitable for X-ray diffraction studies, have been grown at room temperature using 2-methyl-2,4-pentanediol as the precipitant at pH 7.2. The space group of the orthorhombic form is P2(1)2(1)2, with unit cell dimensions a = 133 A, b = 128 A and c = 65 A, and there is one dimeric molecule in the asymmetric unit. The space group of the tetragonal form is P4(2)2(1)2, with unit cell dimensions a = b = 132 A and c = 75 A, with one subunit in the asymmetric unit. The tetragonal crystals diffract to at least 2.0 A resolution.     

Crystallization and preliminary analysis of crystals of cytochrome c2 from Rhodopseudomonas capsulata

Two crystal forms of the cytochrome c2 isolated from Rhodopseudomonas capsulata have been obtained. One crystal form (type I), grown from ammonium sulfate solutions at pH 7.5, belongs to the space group R32 with unit cell dimensions of a = b = 100.0 A, and c = 162.2 A in the hexagonal setting. These crystals most likely contain two molecules in the asymmetric unit. The other crystal form (type II) was obtained from polyethylene glycol 6000 solutions at pH 6.5. Type II crystals belong to the space group P3(1)21 or P3(2)21 with one molecule per asymmetric unit and unit cell dimensions of a = b = 52.4 A, and c = 87.9 A. Both crystal forms diffract to at least 1.8 A resolution and appear to be resistant to radiation damage.     

Crystals of a bovine neurophysin II tripeptide complex

A bovine neurophysin II S-methyl-Cys-Tyr-Phe-NH2 complex has been crystallized using ammonium sulfate as the precipitating agent. The crystals are orthorhombic, the space group is either I222 or I2(1)2(1)2(1) with a = 124.9 A, b = 69.6 A and c = 151.5 A. The crystals diffract to at least 3.0 A resolution. Based on one neurophysin tetramer per asymmetric unit, the Matthews coefficient is calculated to be 3.92 with a solvent content of 69%.     

Single crystals of chitosan

Lamellar single crystals of chitosan were prepared at 125 degrees C by adding ammonia to a low DP fraction of chitosan dissolved in water. The crystals gave sharp electron diffraction diagrams which could be indexed in an orthorhombic P2(1)2(1)2(1) unit cell with a = 8.07 A, b = 8.44 A, c = 10.34 A. The unit cell contained two anti-parallel chitosan chains and no water molecules. It was found that cellulose microfibrils from Valonia ventricosa could act as nuclei for inducing the crystallization of chitosan on cellulose. This produced a shish-kebab morphology.     

Crystallization and preliminary X-ray diffraction studies of human salivary alpha-amylase.

Nonglycosylated alpha-amylase, a major component of human parotid saliva, has been crystallized by the vapor diffusion technique using 2-methyl-2,4-pentanediol as the precipitant in the presence of CaCl2 at pH 9.0. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions of a = 53.3, b = 75.8, and c = 138.1 A. The asymmetric unit contains one amylase molecule. The solvent content is 54%. The crystals are stable to X-rays and diffract up to 2.8 A and appear to be suitable for X-ray diffraction studies.     

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.     

Preliminary crystallographic study of glucose-6-phosphate dehydrogenase from rat liver

Crystals of D-glucose-6-phosphate: NADP+ oxidoreductase were obtained with the hanging drop, vapor diffusion and batch methods from ammonium sulfate-containing solutions. X-ray diffraction photographs indicate that the crystals belong to the orthorhombic space groups I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 66.0 A, b = 140.8 A and c = 177.8 A. These data, together with results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crystal density experiments, indicate that there is one 116,000 Mr dimer per asymmetric unit. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination.