Characterization of single crystals of the large ribosomal particles from Bacillus stearothermophilus

Single, three-dimensional crystals of the 50 S ribosomal subunit from Bacillus stearothermophilus (strain NCA) have been characterized using a synchrotron X-ray source. The crystals are orthorhombic with unit cell dimensions: a = 350 A, b = 670 A, c = 905 A, and contain at least one 2-fold screw axis. With cooling to -2 degrees C, the large crystals (1.0 mm X 0.2 mm X 0.1 mm) diffract to 15 to 18 A resolution and are stable in the synchrotron beam for several hours. Despite the large cell dimensions, the reflections are readily resolved when the X-ray diffraction patterns are densitometered with a 25 microns faster.     

Crystallization and preliminary X-ray studies of V(1)-ATPase of Thermus thermophilus HB8 complexed with Mg-ADP

Crystals have been grown of the V(1)-ATPase sector of the V-type ATP synthase complex (V(0)V(1)) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 A in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a = b = 89.0 A, c = 179.2 A, and gamma = 120 degrees. The unit cell presumably contains one molecule of V(1)-ATPase and the V(m) value is calculated as 3.0 A(3)/Da.     

Preliminary investigation of the phage phi X174 crystal structure

Crystals of the single-stranded DNA bacteriophage phi X174 have been grown. They have a monoclinic unit cell with space group P2(1), unit cell dimensions of a = 306.0 (+/- 0.2) A, b = 361.1 (+/- 0.2) A, c = 299.7 (+/- 0.2 degrees) A, beta = 92.91 degrees (+/- 0.02 degrees) and diffract to at least 2.7 A resolution. There are two virus particles per unit cell. Packing considerations show that the mean diameter of the virus particles is 280 A. The virus separates into two bands in a sucrose gradient. The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles. Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes.     

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 diffraction studies of 5 S rRNA from the thermophilic bacterium Thermus flavus

Crystals of purified 5 S rRNA from Thermus flavus have been obtained. The crystals diffract up to 8 A resolution, using synchrotron radiation, and have the monoclinic space-group C2. The unit cell has the dimensions a = 190 A, b = 110 A, c = 138 A and beta = 117 degrees. The cell volume suggests the presence of four 5 S rRNA molecules per asymmetric unit.     

Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase.

Porphobilinogen deaminase, the polymerase that catalyses the synthesis of preuroporphyrinogen, the linear tetrapyrrole precursor of uroporphyrinogen III, has been crystallized from sodium acetate buffer with polyethylene glycol 6000 as precipitant. The crystals are orthorhombic and the space group is P2(1)2(1)2, with unit cell dimensions a = 88.01 A, b = 75.86 A, c = 50.53 A and alpha = beta = gamma = 90 degrees, indicating a single molecule of 34 kDa in the asymmetric unit. The crystals grow to dimensions of 1 mm x 2 mm x 0.5 mm within two weeks in the dark and are stable in the X-ray beam for at least 40 hours. Diffraction data beyond 1.7 A resolution, observed with a synchrotron radiation source, indicate that a high resolution structure analysis is feasible.     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.     

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.     

Crystallization and preliminary X-ray crystallographic analysis of spruce budworm antifreeze protein.

Antifreeze proteins have the ability to bind to ice with high affinity and inhibit further crystal growth. The insect antifreeze protein from spruce budworm exhibits very high thermal hysteresis activity and is implicated in the protection of overwintering larvae from freezing. This protein has been crystallized in 20-25% polyethylene glycol (Mr 6000), 0.4 M NaCl, 0.1 M Tris-HCl, pH 8.5, by vapor diffusion using the hanging drop method. The resulting crystals are very thin (typically <0.01 mm in the shortest dimension), and only after repeated seeding could crystals be grown large enough for data collection using synchrotron radiation. The crystals belong to the monoclinic space group C2, with cell dimensions a = 82.28 A, b = 62.29 A, c = 63.63 A, and beta = 113.7 degrees. Molecules in the asymmetric unit are related by a twofold axis of symmetry with two molecules present. Native data to a resolution of 2.6 A have been collected with 90.3% completeness and a Rsym of 6.9%.     

Preliminary X-ray investigation of 70 S ribosome crystals from Thermus thermophilus

Large three-dimensional crystals of 70 S from Thermus thermophilus have been grown from solutions of 2-methyl-2,4-pentanediol at 4 degrees C and examined in an X-ray synchrotron beam. The space group is P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = 510 A and c = 378 A. The diffraction patterns extend to better than 20 A.     

Crystallization and preliminary crystallographic studies of the FAD domain of corn NADH: nitrate reductase.

Crystals of the flavin domain of corn nitrate reductase expressed in Escherichia coli have been obtained at room temperature, using sodium citrate as precipitant. The crystals diffract to at least 2.5 A resolution at a synchrotron radiation source. Precession photographs show that they belong to the rhombohedral space group R3 with unit cell dimensions a = b = 145.4 A, c = 47.5 A, alpha = beta = 90 degrees and gamma = 120 degrees. There is one subunit per asymmetric unit which gives a packing density of 3.2 A3/Da, indicating a high solvent content in these crystals.     

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 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.     

Growing calmodulin crystals for X-ray diffraction studies at room temperature in 2 days

Crystals of bovine brain calmodulin have been grown using a novel procedure which utilizes a mixture of alcohols as a precipitant. Crystals were grown by vapor diffusion at room temperature in the presence of 15% ethanol, 25% 2-methyl-2,4-pentanediol, 5.0 mM calcium chloride, and 10.0 mM sodium acetate buffer at pH 4.0. Crystals were visible within 4 h and grew to 0.75 X 0.3 X 0.125 mm in 2 days, without seeding. The crystals of calmodulin are isomorphous with those found by W. J. Cook, J. R. Dedman, A. R. Means, and C. E. Bugg (1980, J. Biol. Chem. 255, 8152-8153). The protein crystallizes in space group P1 with unit cell dimensions a = 29.92 A, b = 55.96 A, c = 24.75 A, alpha = 93.81 degrees, beta = 99.24 degrees, and gamma = 88.40 degrees.     

Crystallization and preliminary X-ray crystallographic analysis of lipase from Pseudomonas cepacia.

Large crystals of lipase from Pseudomonas cepacia have been grown at room temperature from solutions containing 2-methyl-2,4-pentanediol and sodium citrate. They grow within two weeks to typical dimensions of 1.0 mm x 0.5 mm x 0.3 mm. The crystals belong to the monoclinic space group P2(1), with unit cell parameters a = 84.91 A, b = 47.33 A, c = 86.00 A, and beta = 116.09 degrees. And they diffract to about 1.6 A upon exposure to synchroton X-rays. X-ray data have been collected to 2.2 A Bragg spacing from a native crystal.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.

The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.     

Characterization of crystals of an intact monoclonal antibody for canine lymphoma

A monoclonal antibody of the subclass IgG2a specific for canine lymphoma cells has been crystallized by vapor diffusion from polyethylene glycol 8000. the crystals, which occasionally measure nearly a millimeter on edge, have been examined by X-ray diffraction. The crystals are of triclinic space group P1 with unit cell parameters of a = 66.39 A, b = 77.34 A, c = 101.42 A, alpha = 87.60 degrees, beta = 92.55 degrees, gamma = 97.54 degrees and cell volume of V = 4.84 x 10(5) A3. There is one entire antibody molecule as the asymmetric unit of the crystals. Three-dimensional X-ray diffraction data have been collected to 2.8 A resolution and a self rotation function calculation shows a pronounced peak indicating at least an approximate non-crystallographic dyad axis.     

A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography.

Highly ordered two-dimensional crystals of cholera toxin B-subunit pentamers have been grown by specific interaction with planar lipid films containing monosialoganglioside GM1. Electron diffractograms of frozen-hydrated crystals show diffraction peaks extending to beyond 4 A, while electron images diffract to 8 A. A two-dimensional projected structure of cholera toxin B-subunit-GM1 complex has been calculated at 9 A resolution by combining electron diffraction and image data. Crystals present an approximate pgg projection symmetry, with unit cell dimensions a = 119(+/- 1) A, b = 123(+/- 1) A, gamma = 90 degrees. Each pentameric assembly presents two concentric rings of electron scattering density, separated by an area of lower density. The outer and inner rings are centered at 25 A and and 11 A from the pentamer centre, respectively. The apparent projected density of the outer ring is larger than that of the inner ring. We propose that the outer and inner density rings correspond respectively to the peripheral beta-sheet arrangement and the central alpha-helix barrel, recently identified in the crystal structure of the heat-labile enterotoxin from Escherichia coli.