Crystallization and preliminary X-ray diffraction study of cytochrome c552 from Hydrogenobacter thermophilus.

Cytochrome c552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus, exhibits remarkable thermostability. The oxidized cytochrome c552 has been crystallized in an ethanol/water mixture by means of the vapor diffusion method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2, with unit cell dimensions of a = 93.4 A, b = 52.9 A, and c = 32.4 A. Most probably the asymmetric unit contains two molecules of cytochrome c552. The crystals diffract X-rays to better than 2.5 A resolution and are stable to X-ray irradiation.     

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.     

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 of human gelsolin.

Human gelsolin has been crystallized by microdialysis techniques to give single crystals that diffract to 3.5 A resolution. The crystals belong to space group P42(1)2 and have cell dimensions a = 175.0 A, c = 151.6 A. They contain two gelsolin molecules in the asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of a 40 kDa calcium binding protein specifically expressed in plasmodia of Physarum polycephalum.

A calcium binding protein with a molecular mass of 40 kDa (CBP40), the gene product of plasmodial-specific LAV1-2 of Physarum polycephalum, was crystallized in the presence of EDTA. The crystals diffracted X-rays up to a resolution of 3.0 A. They belonged to the trigonal space group, P3221 (or P3121), with unit cell dimensions of a = b = 64.4 A and c = 207.2 A. Ca2+-bound crystals were obtained by soaking in a CaCl2 solution, which gave diffraction data of similar quality. The Ca2+-soaked crystals belonged to the same space group as those crystallized in the presence of EDTA with unit cell dimensions of a = b = 64.4 A and c = 209.4 A.     

Crystallization and preliminary X-ray diffraction analysis of staphylococcal enterotoxin type C.

The Type C staphylococcal enterotoxin produced by Staphylococcus aureus strain FRI-909 has been crystallized using a combination of two precipitants, ammonium sulfate and polyethylene glycol 400, with the addition of small amounts of detergent. Two related crystal forms have been obtained, one triclinic, and one tetragonal, both with one toxin molecule per asymmetric unit. These crystals are stable for at least 75 hr in the X-ray beam and diffract to at least 2.2 and 2.6 A, respectively. The triclinic crystals have unit cell parameters a = 38.5 A, b = 43.7 A, c = 36.9 A, and interaxial angles alpha = 99.9 degrees, beta = 95.8 degrees, and gamma = 98.5 degrees. The tetragonal crystals are of space group P4(1)22 with unit cell parameters a = 43.4 A and c = 278.0 A.     

Crystallization and preliminary crystallographic study of neuraminidase from Micromonospora viridifaciens.

Single crystals of neuraminidase from the bacterium Micromonospora viridifaciens were obtained using the hanging drop vapour diffusion method and polyethylene glycol as precipitant at pH 5.0 or 5.5. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 48.14 A, b = 82.73 A, c = 84.75 A and with one molecule in the asymmetric unit. Diffraction extends to at least 1.7 A.     

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 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.     

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.     

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.     

Two crystal forms of Azotobacter ferredoxin.

Two crystal forms of Azotobacter vinelandii (4Fe-4s)2 ferredoxin I (Fd I) have been grown which are suitable for high resolution x-ray diffraction studies. Tetragonal crystals grow as square bipyramids from ammonium sulfate and Tris buffer using a temperature gradient. The space group is P41212 (or P43212) with a = 55.3, c = 95.9 A and 1 molecule/asymmetric unit. Triclinic crystals grow as plates or laths from ammonium sulfate and phosphate buffer at constant temperature. The space group is P1 with a = 46.8, b = 58.7, c = 64.3 A, alpha = = 105 degrees 05 min, beta = 82 degrees 30 min, gamma = 110 degrees 30 min and 4 or 5 molecules/unit cell. Both crystal forms are stable to x-ray irradiation and diffract beyond 3.0 A resolution.     

Crystallization and preliminary X-ray analysis of phosphoserine aminotransferase from Bacillus circulans subsp. alkalophilus.

Recombinant phosphoserine aminotransferase (EC 2.6.1.52) from Bacillus circulans subsp. alkalophilus was crystallized at room temperature from 0.1 M sodium acetate buffer, pH 4.6, and 2% PEG 20000, using macroseeding techniques. The crystals diffract X-rays to at least 2.0 A nominal resolution. They belong to space group C2 with unit cell dimensions a = 93.2 A, b = 93.1 A, c = 45.6 A, alpha = 90.0 degrees, beta = 106.8 degrees, gamma = 90.0 degrees. A native data set to 2.3 A has been collected. Assuming an average packing density of the crystals, there is one monomer in the asymmetric unit, resulting in a calculated solvent content of 48.2%.     

Preliminary crystallographic data on monomeric and dimeric hemoglobins from the sea cucumber, Molpadia arenicola.

Large single crystals of two distinct globin chains from coelomic cells of the sea cucumber Molpadia arenicola have been prepared and examined by x-ray crystallography. These hemoglobins exhibit a variety of ligand-dependent association states with the met-hemoglobins existing as monomers and liganded hemoglobins as dimers at physiological concentrations. Monomeric methemoglobin C chain crystallizes in space group P21, with a = 46.0 A, b = 45.3 A, c = 40.9 A, beta = 104.5 degrees, and one monomer per asymmetric unit. These crystals exhibit unusual spectroscopic behavior when examined with a polarizer, turning colorless in certain orientations. This implies that all the heme rings are nearly parallel within the crystals. Dimeric cyanmethemoglobin D chain crystallizes in space group P41212 (P43212), with a = b = 77.0 A, c = 61.5 A, and one-half a dimer per asymmetric unit. These homodimers thus possess a molecular 2-fold which is aligned with the crystallographic dyad.     

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.     

Crystallization and preliminary X-ray diffraction studies of pancreatic spasmolytic polypeptide.

Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapour diffusion method. The crystals belong to the space group I222 or I2(1)2(1)2(1) with cell dimensions a = 181.9 A, b = 54.5 A, c = 72.9 A. The crystals diffract to at least 2.5 A resolution and the asymmetric unit contains two molecules (Vm = 3.9 A3/Da) with a solvent content of 68% as determined by density measurements of the crystals. The self-rotation function suggests that the two molecules within the asymmetric unit are related by a 2-fold axis at either 30 degrees or 60 degrees from a in a plane perpendicular to the b axis.     

Crystallization of fragment D from human fibrinogen.

Fragment D from human fibrinogen has been crystallized. The fragment, which is composed of three disulfide-linked chains (alpha' beta' gamma' = 88,000), was generated with either plasmin or mild trypsin digestion. The crystals diffracted out to 3.5 A; the space group is P2(1), unit cell dimensions a = 108 A, b = 48 A, c = 167 A, beta = 106 degrees. Fragment D was also co-crystallized with the ligand GPRP-amide, in which case the space group is consistent with P212121, unit cell dimensions a = 476 A, b = 82 A, c = 432 A.     

Co-crystals of gramicidin A and phospholipid. A system for studying the structure of a transmembrane channel.

Single crystals of a complex of gramicidin A, a transmembrane channel-forming polypeptide, and dipalmitoyl phosphatidylcholine, a phospholipid, have been prepared and characterized by X-ray diffraction. They belong to space group P222(1), with unit cell dimensions a = 26.8 A, b = 27.5 A, c = 32.8 A. The asymmetric unit appears to be a complex of one gramicidin monomer and two phospholipid molecules. The unit cell dimensions, space group, and chemical composition are compatible with lipids packing in a bilayer-like motif, and an end-to-end association of gramicidin monomers to form a functional dimeric unit. The crystals diffract to 2 A and are suitable for structural studies by single-crystal X-ray analysis. This represents the first example of a well-ordered crystalline channel complexed with lipids, and solution of its structure may give insight into mechanisms of ion transport across membranes.     

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%.