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 of the A alpha subunit of protein phosphatase 2A.

The A alpha subunit of human protein phosphatase 2A forms crystals in space group P2(1) with cell dimensions a = 104.0, b = 174.9, c = 168.2 A, and beta angle = 90.2 degrees. At cryogenic temperatures, the crystals diffracted to a resolution limit of approximately 3.0 A. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 A and merged to provide a set that is 93% complete, with Rsym of 9.9%.     

Crystalline beta-cyclodextrin.12H2O reversibly dehydrates to beta-cyclodextrin.10.5 H2O under ambient conditions.

In contact with mother liquor, crystalline beta-cyclodextrin (beta-CD) hydrate has composition approximately beta-CD.12H2O. If crystals are dried at ambient conditions (18 degrees C, approximately 50% humidity), the unit cell volume diminishes approximately 30 to 50 A3. X-ray structure analysis of a dry crystal (0.89 A resolution, 4617 data, R = 0.059) showed the composition beta-CD.10.5 H2O, with approximately 5.5 water molecules in the beta-CD cavity (7 partially and 2 fully occupied sites) and approximately 5.0 between the beta-CD molecules. The positions of the beta-CD host and of most of the hydration waters are conserved during dehydration, but the occupancies of the waters in the beta-CD cavity diminish. Dry crystals put into solvent re-hydrate to the original form. The mechanism of de- and re-hydration is not evident.     

Molecular symmetry of fructose-1,6-diphosphatase by X-ray diffraction analysis.

Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry.     

Preliminary crystallographic study of an L-asparaginase from Vibrio succinogenes

Crystals of an L-asparaginase from Vibrio succinogenes were obtained with the hanging drop method from ammonium sulphate-containing solutions. The crystals belong to the orthorhombic space group P22(1)2(1) with unit cell dimensions of a = 71.3 A, b = 85.8 A, c = 114.0 A, and contain two tetrameric enzyme molecules per unit cell. There are two subunits in the asymmetric unit; a molecular dyad is coincident with the crystallographic dyad. The crystal lattice is similar to that reported for an Escherichia coli asparaginase. Rotation function calculations have revealed that the V. succinogenes enzyme has 222 point group symmetry in the crystal. The second and third molecular dyads differ, however, from the corresponding E. coli asparaginase dyads by approximately 40 degrees. The crystals diffract to at least 2.2 A resolution and are suitable for X-ray crystallographic structure determination.     

Crystallization and preliminary X-ray data for the exocellular beta-lactamase of Bacillus licheniformis 749/C

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is P21, with unit cell dimensions a = 66.77 A, b = 93.77 A, c = 43.57 A and beta = 104.5 degrees. The asymmetric unit consists of two molecules of 28,500 Mr each. The crystals are suitable for structure analysis to at least 2 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.     

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.     

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli.

The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentration approximately 20 microM and approximately 10 microM, respectively. The levels do not change appreciably following heat shock (44 degrees C), but a small increase in Hsc20 is observed following a shift to 10 degrees C. Purified Hsc66 exhibits a low intrinsic ATPase activity (approximately 0.6 min-1 at 37 degrees C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration approximately 5 microM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions.     

Crystallization and preliminary X-ray crystallographic studies on the herpes simplex virus 1 single-stranded DNA binding protein

Crystals of a 60-amino-acid C-terminal deletion mutant of the herpes simplex virus 1 single-stranded DNA binding protein, ICP8, have been grown by hanging drop vapor diffusion. The colorless crystals grow as thin plates to a maximum size of approximately 0.3 mm x 0.3 mm x 0.05 mm. The space group is P2(1)2(1)2(1) with unit cell constants a = 101.2 A, b = 145.8 A, and c = 162.9 A. There are one or two molecules of ICP8 per asymmetric unit.     

Crystallization and molecular symmetry of ornithine decarboxylase from Lactobacillus 30a

Ornithine decarboxylase from Lactobacillus 30a is representative of the large subunit (80 kDa), oligomeric, pyridoxal phosphate-dependent amino-acid decarboxylases. Yellow crystals of ornithine decarboxylase are obtained from polyethylene glycol solutions and belong to space group P6 with unit cell constants a = b = 194.9 and c = 97.44 A, alpha = beta = 90 degrees and gamma = 120 degrees, V = 3.21 x 10(6) A3. Still photographs show reflections at better than 2.4-A resolution. Electron micrographs reported by Guirard and Snell (Guirard, B.M., and Snell, E.E. (1980) J. Biol. Chem. 255, 5960-5964) reveal that the ornithine decarboxylase dodecamer is a hexagonally shaped particle with a point-to-point distance of approximately 210 A and a thickness of approximately 70 A. The crystallographic unit cell can thus accommodate one 10(6)-Da dodecamer (Vm = 3.2 A3/Da), implying that a dimer occupies an asymmetric unit. Tanaka rotation function analysis, using native data (5-7 A) collected from three crystals, reveals that the particle has the expected 622 molecular symmetry with molecular 2-fold axes lying at 20 degrees and 50 degrees from a in the a-b plane. A search for suitable heavy atom derivatives is underway.     

Preliminary crystallographic study of pyrimidine dimer-specific excision-repair enzyme from bacteriophage T4

Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2(1), with unit cell parameters: a = 41.4 A, b = 40.1 A, c = 37.5 A, beta = 90.01 degrees. The unit cell contains two 16,000 Mr molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.     

Preliminary X-ray crystallographic study of lysozyme produced by Streptomyces globisporus

Lysozyme from Streptomyces globisporus has been crystallized in a form suitable for X-ray structure analysis using ammonium sulfate as a precipitant. The crystals are hexagonal, space group P6(1)22 (P6(5)22) with unit cell dimensions: a = b = 129 A, c = 143 A. There are three or four molecules per asymmetric unit. The crystals diffract X-rays to at least 3.0 A resolution.     

Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae.

Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.     

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.     

Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein

The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa. The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet). The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98%. Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit. The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination.     

Crystallization and preliminary X-ray analysis of complexes of peptide inhibitors with human recombinant and mouse submandibular renins.

Inhibitor-complexed crystals of mouse and human renins suitable for X-ray analysis have been prepared. The mouse renin is complexed with a non-hydrolysable decapeptide analogue of rat angiotensinogen containing a hydroxyethylene isostere in place of the scissile bond. The crystals are monoclinic, space group P2(1) with cell dimensions a = 78.3 A, b = 117.8 A, c = 85.9 A, beta = 101.18 degrees containing four molecules per asymmetric unit. The human renin is fully glycosylated and complexed with a tetrapeptide containing norstatine. The complex crystallises in the cubic space group P2(1)3 with a = 143.1 A and has two molecules in the asymmetric unit. The rotation function of the mouse renin complex indicates pseudo 222 symmetry while that of human renin indicates a pseudo 2-fold axis. Full structural analyses of the two complexes are underway.     

Crystallization and preliminary analysis of crystals of high potential iron-sulfur protein from Rhodospirillum tenue

Large single crystals of the high potential iron-sulfur protein isolated from Rhodospirillum tenue strain 3761 have been obtained. They belong to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A, and beta = 90.8 degrees. There are two molecules in the asymmetric unit. Based on oscillation photographs, the crystals diffract to at least 1.6 A resolution. They are stable in the x-ray beam and appear suitable for a high resolution x-ray structure analysis.     

Structure of tropomyosin at 9 angstroms resolution.

We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.