Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.     

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.     

Crystallization and preliminary X-ray diffraction studies of the engrailed homeodomain and of an engrailed homeodomain/DNA complex

The homeodomain from the engrailed protein of Drosophila has been crystallized from ammonium phosphate at pH 6.8. The crystals form in space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 44.8 A and c = 118.2 A. These crystals diffract to 1.8 A resolution. A complex containing the engrailed homeodomain and a duplex DNA site also has been crystallized. The cocrystals form in space group C2 with a = 131.2 A, b = 45.5 A, c = 72.9 A and beta = 119.0 degrees. These crystals diffract to 2.6 A resolution.     

Crystallization of a macromolecular ring assembly of tubulin liganded with the anti-mitotic drug podophyllotoxin

The interaction of the anti-cancer drug podophyllotoxin with a high-molecular-weight assembly of tubulin has been employed to produce three-dimensional crystals from avian erythrocyte tubulin as well as from pig brain tubulin. Avian erythrocyte tubulin crystals belong to the space group C2 with unit cell dimensions a = 740 A, b = 330 A, c = 460 A, beta = 128 degrees. The basis of these crystals is ring oligomers with a molecular mass of approximately 6 x 10(6) Da. So far, the crystals diffract to 8-A resolution and a first complete data set to 12-A resolution has been collected under cryogenic conditions. The crystals grew from conventionally purified tubulin consisting of multiple isoforms and different posttranslational modifications. Thus, the use of highly homogeneous tubulin preparations should improve the diffraction quality of these crystals.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli

Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method. The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees. There are three subunits in the asymmetric unit. The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies.     

Preparation of crystals of chicken cytosolic aspartate amino- transferase, suitable for high resolution x-ray structural analysis

The crystals of cytosolic chicken aspartate aminotransferase were grown from polyethylene glycol solutions. Two of the four crystal modifications obtained diffract to 1.8 A resolution. The crystals of the free holoenzyme belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 56.9, b = 126.9, c = 124.6 A. The crystals of the enzyme-maleate complex belong to the same space group with slightly different unit cell dimensions of a = 56.5, b = 126.1, c = 124.6 A. The influence of ions of several divalent metals, dioxane and non-ionic detergent beta-octylglucoside on crystallization have been investigated. The best crystals were obtained in the presence of Mg2+ ions. These crystals were used for data collection on the diffractometer.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

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.     

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 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 diffraction studies of malate dehydrogenase from Streptomyces aureofaciens

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C222(1) with unit-cell parameters a = 53.2 A, b = 104.6 A, c = 520.0 A, alpha = beta = gamma = 90( degrees ), MDH-NADH crystals to space group C2 with unit-cell parameters a = 51.5 A, b = 51.5 A, c = 256 A, alpha = beta = gamma = 90( degrees ), and MDH-NADPH crystals to space group C222(1) with unit-cell parameters a = 72, A b = 72 A, c = 520 A, alpha = beta = gamma = 90( degrees ). The crystal of native MDH diffracted to 2.1 A resolution.     

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.     

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.     

Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1

The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of beta-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 degrees C. The orthorhombic crystals (space group P2(1)2(1)2(1)) have cell constants a = 142.5 A, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.     

Crystallization and preliminary crystallographic study of bacterial alpha-amylases

Crystallization of bacterial alpha-amylases has been achieved by the hanging-drop vapor diffusion method. The crystals of Bacillus licheniformis and B. licheniformis 584 amylases are isomorphous to each other. The crystals of B. licheniformis amylase belong to the tetragonal system, space group P4(2)2(1)2 with cell dimensions of a = 119.3 and c = 85.4 A. The asymmetric unit contains one molecule of amylase, with a volume per molecular mass, Vm, of 2.75 A3/Da. The crystals of B. licheniformis and B. licheniformis 584 amylases diffract beyond 2.5 A resolution and are suitable for X-ray diffraction analysis. The crystals of B. amyloliquefaciens amylase are orthorhombic, and have space group C222(1), with cell dimensions of a = 154, b = 298, and c = 90 A. The asymmetric unit contains three to five molecules. In the crystallization of B. licheniformis and B. licheniformis 584 amylases, the addition of EDTA was indispensable to obtain large single crystals, while it had an adverse effect on the crystallization of B. amyloliquefaciens amylase, producing a large amount of small crystals.     

Crystallization of and preliminary crystallographic data for allosteric L-lactate dehydrogenase from Bifidobacterium longum

L-Lactate dehydrogenase from Bifidobacterium longum aM101-2 was overexpressed in Escherichia coli and then purified. The enzyme was crystallized from a polyethylene glycol 6000 solution by the hanging drop vapor diffusion method. Crystals grown in the presence of NADH (type II), both NADH and oxamate (type III), and NADH, oxamate, and FBP (type IV) were analyzed. All three crystal forms belong to the orthorhombic system, space group P2(1)2(1)2. The cell dimensions of the type II crystals were a = 106.2 A, b = 131.6 A, and c = 63.8 A. Those of the type III and type IV crystals were a = 106.4 A, b = 131.4 A, and c = 63.8 A. The type III crystals diffract X-rays to beyond 2.5 A spacing. The type II and type III crystals were stable as to X-ray irradiation.     

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.