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 malate dehydrogenase from Streptomyces aureofaciens

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C222(1) with unit-cell parameters a = 53.2 A, b = 104.6 A, c = 520.0 A, alpha = beta = gamma = 90( degrees ), MDH-NADH crystals to space group C2 with unit-cell parameters a = 51.5 A, b = 51.5 A, c = 256 A, alpha = beta = gamma = 90( degrees ), and MDH-NADPH crystals to space group C222(1) with unit-cell parameters a = 72, A b = 72 A, c = 520 A, alpha = beta = gamma = 90( degrees ). The crystal of native MDH diffracted to 2.1 A resolution.     

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.     

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.     

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 murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

X-ray studies on triclinic crystals of fatty acid binding protein. Examples of an extremely X-ray-resistant protein

Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 A, b = 39.4 A, c = 30.6 A, alpha = 113.6 degrees, beta = 113.8 degrees, gamma = 88.8 degrees. Crystal diffraction extends to at least 2.25 A resolution and the crystals are stable in the X-ray beam for more than 450 h. One native data set to 2.5 A resolution has been collected.     

Single crystals of large ribosomal particles from Halobacterium marismortui diffract to 6 A

Large, well-ordered three-dimensional crystals of 50 S ribosomal subunits from Halobacterium marismortui have been obtained by seeding. The crystals have been characterized with synchrotron X-ray radiation as monoclinic, space group P2(1), with unit cell dimensions of a = 182(+/- 5) A, b = 584(+/- 10) A, c = 186(+/- 5) A, beta = 109 degrees. At 4 degrees C, the crystals (0.6 mm X 0.6 mm X 0.1 mm) diffract to 6 A resolution and are stable in the synchrotron beam for several hours. Compact packing is reflected from the crystallographic unit cell parameters and from electron micrographs of positively stained thin sections of embedded crystals.     

Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.

A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.     

Preliminary X-ray crystallographic study of methanol dehydrogenase from Methylophilus methylotrophus

Single crystals of methanol dehydrogenase from Methylophilus methylotrophus have been prepared by the macroseeding method. The crystals belong to the monoclinic space group C2, and have unit cell parameters a = 125.62 A, b = 63.83 A, c = 83.99 A, and beta = 93.24 degrees. There is one 62,000 Mr monomer in the asymmetric unit. The crystals diffract to beyond 2.0 A resolution.     

Preliminary crystallographic data for stearoyl-acyl carrier protein desaturase from castor seed.

Recombinant stearoyl-acyl carrier protein desaturase (EC 1.14.99.6) from castor seed has been crystallized with polyethylene glycol 8000 as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 81.3, b = 146.4 and c = 197.7 A. The observed diffraction pattern extends to at least 2.5 A resolution. Rotation function calculations indicate a non-crystallographic 3-fold rotation axis parallel to the crystallographic a-axis. Perpendicular to this axis, 2-fold rotation axes were found at 30 degrees intervals, i.e. maxima at kappa = 180 degrees, phi = 90 degrees and omega = 30 degrees and 60 degrees, respectively. Together with the packing density of the crystals (Vm = 2.4 A3/Da for n = 6), these results suggest, that the crystal asymmetric unit most likely contains a hexamer of desaturase subunits.     

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%.     

Crystallization and preliminary X-ray investigation of factor D of human complement

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 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.     

Crystallization and preliminary X-ray diffraction analysis of catalase HPII from Escherichia coli

Green crystals of the hexameric catalase HPII from Escherichia coli have been obtained by the hanging-drop method. The crystals belong to the monoclinic space group P2 with a = 123 A, b = 132 A, c = 93 A, beta = 112.5 degrees. There are three subunits in the asymmetric unit. The crystals diffract at least to 3.2 A resolution and are suitable for further X-ray diffraction studies.     

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.     

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.     

Single crystals of bacteriophage T7 RNA polymerase

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.     

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.     

Characterization of crystals of an Fab fragment of a murine monoclonal antibody.

The Fab fragment of an antibody, made against an E2-specific feline infectious peritonitis virus neutralizing antibody, has been crystallized in a form suitable for X-ray diffraction analysis from PEG 4000 using vapor diffusion methods. The Fab fragment crystals diffract to about 2.9 A resolution and are of triclinic space group P1. Unit cell dimensions, by which the reciprocal lattice can be indexed, are a = 57.16 A, b = 70.85 A, c = 75.81 A, alpha = 85.11 degrees, beta = 121.28 degrees and gamma = 116.33 degrees. There are two Fab fragments comprising the asymmetric unit of the crystals. The presence of a pseudo-mirror plane in the diffraction pattern suggests the presence of at least an approximate dyad axis relating the two Fab fragments within the asymmetric unit.