Expression, purification, and crystallization of natural and selenomethionyl recombinant ribonuclease H from Escherichia coli

Ribonuclease H (RNase H) from Escherichia coli is an endonuclease that specifically degrades the RNAs of RNA:DNA hybrids. The enzyme is a single polypeptide chain of 155 amino acid residues, of which 4 are methionines. To solve the crystallographic three-dimensional structure of E. coli RNase H by the multi-wavelength anomalous diffraction technique, we have constructed methionine auxotrophic strains of E. coli that overexpress selenomethionyl RNase H. MIC88 yields about 10 mg of selenomethionyl RNase H per liter of culture, which is comparable to the overexpression of the natural recombinant protein. We have purified both proteins to homogeneity and crystallized them isomorphously in the presence of sulfate. These are Type I crystals of space group P2(1)2(1)2(1) with the cell parameters a = 41.8 A, b = 86.4 A, c = 36.4 A, one monomer per asymmetric unit, and approximately 36% (v/v) solvent. Crystals of both proteins diffract to beyond 2-A Bragg spacings and are relatively durable in an x-ray beam. On replacement of sulfate with NaCl, crystals of natural RNase H grow as Type I' (very similar to Type I) at pH between 7.0 and 8.0; at pH 8.8, crystals of Type II are obtained in space group P2(1)2(1)2(1) with a = 44.3 A, b = 87.3 A, and c = 35.7 A. Type II crystals can be converted to Type I by soaking in phosphate buffer. RNase H crystals of Type II have also been reported by Kanaya et al. (Kanaya, S., Kohara, A., Miyakawa, M., Matsuzaki, T., Morikawa, K., and Ikehara, M. (1989) J. Biol. Chem. 264, 11546-11549).     

Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase.

A thermostable mutant of kanamycin nucleotidyltransferase isolated by cloning and selection for kanamycin resistance in Bacillus stearothermophilus at 70 degrees C has been crystallized in a form suitable for high-resolution diffraction analysis. This enzyme catalyzes nucleotidyl group transfer from nucleoside triphosphates such as ATP to hydroxyl groups of various aminoglycosides, thus inactivating the antibiotic. The kanamycin nucleotidyltransferase gene, originally encoded on plasmid pUB110 from the mesophile Staphylococcus aureus, was transferred to the thermophile B. stearothermophilus via shuttle plasmids and the mutant carrying the substitutions D80Y and T130K was isolated from kanamycin-resistant colonies grown at 70 degrees C. The thermostable enzyme was crystallized in two forms from solutions of polyethylene glycol 8000 (PEG8000) using batch and vapor diffusion methods. Type I crystals grown from 19% (w/v) PEG8000 and 200 mM NaCl belong to the orthorhombic space group C222(1), have unit cell dimensions of a = 128.4, b = 156.8, c = 155.8 A, and diffract to at least 2.4-A resolution. The type II form of the crystals were grown from 10% PEG8000, 200 mM KCl, and 3 mM CoCl2, and belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 78.9, and c = 220.4 A; these crystals diffract to at least 2.5-A resolution.     

Human recombinant factor XIII from Saccharomyces cerevisiae. Crystallization and preliminary x-ray data

Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8. The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A. The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa. A 3.5-A resolution data set for the native protein has been collected. Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-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.     

Crystallization and preliminary X-ray investigation of a recombinant form of human gamma-interferon

A genetically engineered human gamma-interferon has been crystallized from solutions of ammonium sulfate. The crystals are trigonal, space group R32, with hexagonal axes of a = 114.0(1) A and c = 314.9(2) A. The crystals are moderately stable to x-rays and diffract to 2.85-A resolution. Density measurements indicate that the crystallographic asymmetric volume contains four interferon molecules.     

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.     

Crystallization and preliminary x-ray characterization of bovine growth hormone. Purification of bovine prolactin and growth hormone

A new purification scheme for both prolactin and growth hormone from bovine pituitaries has been developed which avoids the use of potentially damaging solution conditions. Both hormones were greater than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had specific activities similar to or greater than standard samples of the same hormone as judged by several bioassays. Small single crystals of bovine growth hormone were obtained by vapor diffusion techniques. Examination of these crystals by x-ray diffraction, using the Cornell High Energy Synchrotron Source, showed that they were well ordered, and exhibited diffraction to 2.8-A resolution on still photographs. Precession and oscillation photographs showed that they belonged to the orthorhombic space group P2(1)2(1)2(1) (or P2(1)2(1)2) with unit cell dimensions a = 219 A, b = 51.9 A, c = 68.9 A. The density of the crystals was 1.19 +/- 0.02 g/ml from which the presence of eight 45,000-dalton dimers/unit cell was deduced. The protein content of the crystals was shown by isoelectric focusing to be identical to that of purified growth hormone in solution. These crystals appear suitable for use in the x-ray structure determination of bovine growth hormone to at least 3.2-A resolution.     

Crystallization of and preliminary X-ray data for bovine carbonic anhydrase III

Crystals of bovine carbonic anhydrase III have been grown in a solution of polyethylene glycol. The crystals are monoclinic, space group P2(1), with the unit cell parameters a = 50.6 A, b = 44.7 A, c = 56.9 A, and beta = 90.3 degrees. The asymmetric unit contains 1 molecule. The diffraction pattern extends beyond 2.0-A resolution.     

Preliminary X-ray diffraction analysis of human chorionic gonadotropin

Hexagonal bipyramidal crystals of deglycosylated human chorionic gonadotropin have been grown using the method of vapor diffusion against ammonium sulfate. These crystals grow to nearly 0.4 mm along each axis, diffract to better than 3.5-A resolution and are relatively stable to irradiation. The crystals belong to the hexagonal space group P6(1)22 or enantiomer, and have unit cell parameters a = b = 88.7 A and c = 177.3 A.     

Crystallization and preliminary X-ray studies of pyridoxal kinase from sheep brain

Crystals of pyridoxal kinase from sheep brain have been grown from solutions of ammonium sulfate or Na+/K+ phosphate. The crystals are trigonal, space group P3(1)21 with axes a = b = 102.2 A and c = 58.5 A. The crystals are quite stable to x-rays and diffract at 2.2-A resolution. This macromolecule is a 80,000-kDa dimer and utilizes the 2-fold symmetry of the space group, resulting in one subunit/asymmetric unit.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

Crystallization of the protective antigen protein of Bacillus anthracis

The protective antigen protein, one of the three separate proteins constituting the exotoxin system of Bacillus anthracis, has been crystallized in a form suitable for structural studies. The crystal form which is most amenable to x-ray analysis is orthorhombic, space group P2(1)2(1)2(1), a = 101.1 A, b = 95.4 A, c = 87.3 A, with one protective antigen monomer/asymmetric unit. The crystals diffract to approximately 3.0-A resolution.     

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 immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

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

Crystals of thymidine phosphorylase from Escherichia coli 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 axes are a = 132.0 (1) and c = 67.2 (1) A. The crystals are quite stable to x-rays and diffract beyond 2.6-A resolution. The molecule is a dimer and utilizes the 2-fold symmetry of the space group, resulting in one subunit per asymmetric unit.     

Crystallization and preliminary x-ray investigation of colicin E3 in complex with its immunity protein

Crystals of the colicin E3-immunity protein complex have been grown from solutions of citrate at pH 5.6. The crystals are monoclinic, space group P2(1), with unit cell dimensions a = 67.71, b = 196.67, c = 85.58 A, and beta = 113.67 degrees. The crystals diffract to 3-A resolution and are stable in the x-ray beam for at least a day. Although the stoichiometry of the complex in solution is 1:1 there are two, three, or four such binary complex molecules in the asymmetric unit.     

Crystallization and preliminary diffraction data of Escherichia coli ADP glucose pyrophosphorylase

ADP glucose pyrophosphorylase from Escherichia coli has been crystallized from polyethylene glycol 8000 solutions. The crystals are: orthorhombic, a = 155(2), b = 153(2), c = 174(2) A, space group P2(1)2(1)2(1), four tetrameric molecules/unit cell. This gives a solvent fraction of about 75% consistent with the relatively poor diffraction quality of crystals (5.0-A resolution) and their sensitivity to x-ray exposure damage. Ways of circumventing the former and improving the latter are proposed.     

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.     

Preliminary crystallographic study of bluetongue virus capsid protein, VP7.

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.