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.     

Crystallization of a soluble form of the rat T-cell surface glycoprotein CD4 complexed with Fab from the W3/25 monoclonal antibody

The structure of the T-lymphocyte cell surface glycoprotein CD4 is of considerable biological and medical interest. Recombinant rat CD4 expressed in soluble form in mammalian cells and complexed with W3/25 monoclonal Fab fragments formed crystals that diffract to 3.5 A and have the orthorhombic space group P2(1)2(1)2 or P2(1)2(1)2(1). The unit cell has dimensions a = 317 A, b = 161 A and c = 41.8 A and the asymmetric unit consists of two CD4:Fab complexes. These crystals are of suitable quality for X-ray diffraction analysis.     

Crystallization and preliminary x-ray diffraction studies of two new antigen-antibody (lysozyme-Fab) complexes

The complexes between the Fab fragments of two monoclonal anti-lysozyme antibodies, Fab10.6.6 (high affinity) and D44.2 (lower affinity), and their specific antigen, hen egg-white lysozyme, have been crystallized. The antibodies recognize an antigenic determinant including Arg68, but differ significantly in their association constants for the antigen. Two crystalline forms were obtained for the complex with FabF10.6.6, the higher affinity antibody. One of them is monoclinic, space group P21, with unit cell dimensions a = 145.6 A, b = 78.1 A, c = 63.1 A, beta = 89.05 degrees, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 3 A making this form suitable for high-resolution X-ray diffraction studies. The second form crystallizes in the triclinic space group P1, with unit cell dimensions a = 134.0 A, b = 144.7 A, c = 98.6 A, alpha = 90.30 degrees, beta = 97.1 degrees, gamma = 90.20 degrees, consistent with the presence of 10 to 12 molecules of the complex in the unit cell. These crystals do not diffract X-rays beyond 5 A resolution. The antigen-antibody complex between FabD44.2, the lower affinity antibody, and hen egg-white lysozyme crystallizes in space group P2(1)2(1)2(1), with unit cell dimensions a = 99.7 A, b = 167.3 A, c = 84.7 A, consistent with the presence of two molecules of the complex in the asymmetric unit. These crystals diffract X-rays beyond 2.5 A resolution.     

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.     

Crystal structures of cyclomaltohexaose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol.

Crystal structures of cyclomaltohexose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol have been determined by single-crystal X-ray diffraction. The space group of the alpha-cyclodextrin-p-bromophenol complex is P2(1)2(1)2(1) with unit cell dimensions of a = 15.318(3), b = 24.733(3), c = 13.457(2) A, and that of the alpha-cyclodextrin-m-bromophenol complex is P2(1)2(1)2 with unit cell dimensions of a = 25.858(7), b = 27.263(8), c = 8.145(3) A. In crystals, the alpha-cyclodextrin-p-bromophenol complex and the alpha-cyclodextrin-m-bromophenol complex form a layer-type and a channel-type molecular packing structure, respectively. The intermolecular hydrogen-bond interactions of the hydroxyl groups of bromophenols are closely related to the molecular packing structure.     

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.     

Preliminary crystallographic studies on duck ovotransferrin

Crystals of duck ovotransferrin and duck apo-ovotransferrin have been grown from polyethylene glycol solutions. For both crystals, the space group is P2(1)2(1)2(1), the unit cell dimensions for the ovotransferrin are a = 49.6 A, b = 85.6 A, c = 178.7 A and for the apo-ovotransferrin a = 77.6 A, b = 98.8 A, c = 127.0 A, giving four molecules in the unit cell.     

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.     

Preliminary crystallographic studies of the amino terminal half of human lactoferrin in its iron-saturated and iron-free forms.

The amino terminal half of human lactoferrin (LfN) produced from transfected baby hamster kidney cells has been crystallized in its iron-saturated and iron-free forms. The crystals of glycosylated LfN and deglycosylated LfN are monoclinic, space group C2, with cell dimensions a = 133.0 A, b = 58.3 A, c = 58.3 A, alpha = 90.0 degrees, beta = 114.7 degrees, gamma = 90.0 degrees, and one molecule per asymmetric unit. Crystals of apo LfN have also been prepared using deglycosylated protein. These crystals are tetragonal, space group P4(1)2(1)2 (or P4(3)2(1)2), with cell dimensions of a = b = 58.4 A and c = 217.2 A and one molecule per asymmetric unit. Both the iron-saturated and the iron-free crystals are suitable for high resolution X-ray analysis.     

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 x-ray studies of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with activator and a transition state analogue

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been purified from spinach and crystallized by equilibrium vapor diffusion with polyethylene glycol 6000 as a precipitant. Crystals suitable for x-ray studies were obtained from a binary complex with a transition state analogue, 2-C-carboxy-D-arabinitol 1,5-bisphosphate, and a quaternary complex with 2-C-carboxy-D-arabinitol 1,5-bisphosphate, Mg2+, and HCO-3. Two forms of crystals were obtained in the presence of 2-C-carboxy-D-arabinitol 1,5-bisphosphate. Form B crystals are plates which have orthorhombic space group P2(1)2(1)2 with unit cell dimensions a = 184 A, b = 218 A, and c = 119 A. Form C crystals are tetragonal needles with space group I422 and with cell dimensions a = b = 275 A and c = 178 A. In both forms, the asymmetric unit contains half a molecule.     

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.     

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.     

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 structural studies of neurotrophin-3.

Neurotrophin-3 (NT-3) has been crystallized in 2 forms. Orthorhombic crystals, space group P2(1)2(1)2, diffracted to 2.8 A and have cell dimensions a = 39.1 A, b = 54.0 A, and c = 65.5 A. The second form is space group P4(3)2(1)2, with cell dimensions a = b = 67.1 A, and c = 107.9 A. The tetragonal crystals diffract to 2.8 A at room temperature and 2.5 A at -100 degrees C. The unit cell dimensions change significantly upon freezing, a = b = 66.1 A, and c = 102.8 A. Phases for the orthorhombic form were obtained by molecular replacement using nerve growth factor as the search model. A partially refined model of the NT-3 dimer (75% complete) was then oriented and positioned in the tetragonal cell.     

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.     

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.     

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.     

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 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.