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.     

Preparation, crystallization and preliminary X-ray analysis of the Fab fragment of monoclonal antibody MN423, revealing the structural aspects of Alzheimer's paired helical filaments

Monoclonal antibody (mAb) MN423 recognizes Alzheimer's disease specific conformation of tau protein assembled into paired helical filaments (PHF). Since the three-dimensional structure of PHF is currently unavailable, the structure of MN423 binding site could provide important information about PHF conformation with the consequences for the Alzheimer's disease prevention and cure. Fab fragment of MN423 was prepared and purified. We have identified two different conditions for crystallization of the Fab fragment that yielded two crystal forms. They diffracted to 3.0 and 1.6 A resolution with four and one molecule in the asymmetric unit, respectively. Both crystal forms belonged to the space group P2(1) with unit cell parameters a = 76.4 A, b = 138.4 A, c = 92.4 A, beta = 101.9 degrees , and a = 71.5 A, b = 36.8 A, c = 85.5 A, beta = 113.9 degrees .     

Two crystal structures of the leupeptin-trypsin complex.

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.     

Preparation and X-ray characterization of four new crystal forms of jacalin, a lectin from Artocarpus integrifolia

Four new crystal forms of the anti-T lectin from jackfruit (Artocarpus integrifolia) have been prepared and characterized. Three of them, two monoclinic (P21, a = 59.4 A, b = 83.3 A, c = 63.5 A, beta = 107.7 degrees; C2, a = 106.1 A, b = 53.9 A, c = 128.0 A, beta = 95.0 A) and one orthorhombic (C222(1), a = 98.1 A, b = 67.3 A, c = 95.1 A) were grown with 2-methylpentan-2,4-diol (MPD) as the precipitant while the fourth, an hexagonal form (P6(1)22, a = b = 129.6 A, c = 157.9 A), was obtained in the presence of methyl-alpha-D-galactopyranoside with polyethylene glycol 4000 as the precipitant. The reported relative molecular mass (Mr) of the lectin was found to be inconsistent with the solvent content of the crystals estimated using measured densities. The Mr was redetermined using size-exclusion chromatography in the presence of methyl-alpha-D-galactopyranoside and Ferguson-plot analysis of mobilities in polyacrylamide gel electrophoresis. The redetermined Mr (66,000) is consistent with the measured crystal densities. The orthorhombic and the hexagonal forms, which have one half molecule and one molecule, respectively, in the asymmetric unit, are suitable for high-resolution X-ray analysis.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

Crystallization of a ternary complex of lactate dehydrogenase from Bacillus stearothermophilus

Bacillus stearothermophilus lactate dehydrogenase was purified from an overexpressing Escherichia coli cell line. The enzyme has been crystallized in several different forms. All of these crystal forms were grown in the presence of NADH, sodium oxamate and fructose 1,6-bisphosphate. Three crystal forms have been characterized, an orthorhombic P2(1)2(1)2 (type III, a = 86 A, b = 105 A, c = 136 A) and two monoclinic P21 forms (type IV, a = 85 A, b = 118 A, c = 136 A, beta = 96 degrees; type V, a = 112 A, b = 85 A, c = 136 A, beta = 91 degrees). Precession photographs from these crystal forms are very alike, suggesting the molecular packing to be similar in all three forms. The P21 type IV crystals diffract to beyond 2 A spacing and are stable to irradiation with X-rays. A complete medium-resolution (4.7 A) dataset has been collected from a single crystal using synchrotron radiation. Rotation function studies with these data show the two tetramers of the asymmetric unit to be in very similar orientations. Higher-resolution data are being collected.     

Crystallization and preliminary X-ray studies of the pectate lyase from Bacillus subtilis.

The pectate lyase (EC 4.2.2.9) from Bacillus subtilis has been crystallized. Crystals of form 1, grown by the hanging drop method using polyethylene glycol as precipitant, diffract to at least 2.4 A resolution. They belong to the spacegroup P2(1) with a = 132.9 A, b = 41.2 A, c = 156.8 A and beta = 114.9 degrees with probably four molecules in the asymmetric unit. A second crystal form grown from 2-methyl-2,4-pentandiol also belongs to the spacegroup P2(1) with a = 55.0 A, b = 88.1 A, c = 50.2 A and beta = 109.0 degrees. These crystals diffract to at least 2.0 A and have one molecule in the asymmetric unit. Both crystal forms are suitable for the determination of high-resolution structures.     

Molecular size and symmetry of the bacterioferritin of Escherichia coli. X-ray crystallographic characterization of four crystal forms

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 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 X-ray study of pig lens aldose reductase

Crystals of pig lens aldose reductase have been grown from polyethylene glycol solutions at pH 6.2 and analysed by X-ray diffraction. Two crystal forms were obtained. The first belongs to space group P1 with unit cell dimensions a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees, gamma = 79.0 degrees, with four molecules in the unit cell related by a 222 non-crystallographic symmetry. The second crystal form is hexagonal. The space group is P6(2)22 with a = b = 101 A, c = 257 A and two molecules in the asymmetric unit. Both forms are suitable for X-ray structure analysis to better than 3 A resolution.     

Crystallization and subunit structure of canine myeloperoxidase

Three crystal forms of canine myeloperoxidase are described. An orthorhombic form in space group P2(1)2(1)2(1) has unit cell dimensions: a = 108.3 A (1 A = 0.1 nm) b = 205.9 A and c = 139.9 A. A trigonal form in space group P3(1)21 or P3(2)21 has unit cell dimensions: a = b = 138.9 A and c = 145.2 A. A monoclinic form in space group C2 has unit cell dimensions: a = 117.2 A, b = 96.9 A, c = 131.4 A and beta = 116.3 degrees. Unusual features in the diffraction patterns of the monoclinic form place restrictions on the molecular packing in the crystal. The proposed model for the molecular packing requires that the myeloperoxidase molecule consist of two identical or near-identical halves. In the intact molecule these halves may be related either by a crystallographic dyad axis or by an approximate dyad axis in which one subunit is translated relative to the other by 3.2 A along the symmetry axis. The trigonal crystal form appears most suitable for high-resolution X-ray structural analysis.     

The crystal structure of the 1:1 inclusion complex of beta-cyclodextrin with benzamide

The 1:1 inclusion complex of beta-cyclodextrin and benzamide was prepared and characterized by single crystal X-ray diffraction, PXRD, TGA, and IR. This complex crystallizes in the monoclinic P2(1) space group with unit cell constants a=15.4244(16), b=10.1574(11), c=20.557(2)A, beta=110.074(2) degrees , V=3025.1(6)A(3). The guest molecule projects into the beta-cyclodextrin cavity from the primary hydroxyl side. The amide group protrudes from the primary hydroxyl side and forms hydrogen bonds with the adjacent beta-cyclodextrin molecule. There are six crystallized water molecules, which play crucial roles in crystal packing.     

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.     

Intercalation of water molecules between nucleic acid bases in the crystal structures of 6-azathymine hemihydrate and 5-amino-2-thiocytosine dihydrochloride dihydrate

The crystal structure of 6-azathymine hemihydrate (6AzTH) exhibits a novel intercalation of water molecules interposed half-way between the modified bases 6.3 to 6.7 A apart. The crystal contains four molecules of 6-azathymine (6AzT) and two water molecules as the independent repeating unit. These two water molecules together with the four bases form two separate water sandwiches. In the crystal structure these sandwiches form two sets of local clusters. The anhydrous crystalline form of 6AzT, on the other hand, is stabilized by base stacking interactions. Both the water molecules in 6AzTH that are involved in sandwich formation have trigonal coordination around them. A reexamination of the crystal structure of 5-amino-2-thiocytosine (5A2TC) revealed that one of the water molecules in this structure also forms a water sandwich and has trigonal coordination whereas the other water molecule with tetrahedral coordination does not form a sandwich. The environment and the characteristics of the intercalated water molecule in these structures suggest a possible role for such water intercalations in the dynamics of DNA. Crystals of 6AzTH are monoclinic, space group P21/n, with unit cell parameters a = 8.861 (1), b = 13.177 (3), c = 20.662 (2) A, beta = 93.35 (1) degrees, and Z = 16. From diffractometer data (2503 reflections, greater than or equal to 3 sigma), the crystal structure was solved and refined to an R of 0.056.     

Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide.

Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.     

Crystallization and preliminary diffraction analysis of cholesterol esterase from Candida cylindracea

Cholesterol esterase (EC 3.1.1.13) from the microorganism Candida cylindracea has been crystallized in two forms. Crystals, typically 0.30 x 0.15 x 0.10 mm in size, diffract rotating anode generated x-rays to beyond 3 A are suitable for data collection for an x-ray crystallographic investigation. A monoclinic crystal form in the space group P2(1) was found to have cell dimensions of a = 122.9 A, b = 101.0 A, c = 95.2 A and beta = 108.3 degrees. The asymmetric unit of the cell contains two dimers of 129 kDa each. A second crystal form, in the triclinic space group P1, has cell dimensions of a = 58.6 A, b = 88.7 A, c = 58.6 A, alpha = 93.3 degrees, beta = 113.8 degrees and gamma = 96.0 degrees, and has one dimer per asymmetric unit.     

Preparation,characterization, and antitumor activity of new cisplatin analogues with 1-methyl-4-(methylamino)piperidine: crystal structure of [PtII(1-methyl-4-(methylamino) piperidine)(oxalate)

A series of new platinum(II) complexes of the type [Pt(II)(mmap)X] (where mmap, 1-methyl-4-(methylamino)piperidine and X, 1,1-cyclobutanedicarboxylato (CBDCA), oxalato, malonato, methylmalonato, dimethylmalonato, ethylmalonato, diethylmalonato or 2,3-naphthalene dicarboxylato (NDCA)) have been synthesized and characterized by elemental analysis, infrared (IR), and 13C and 195Pt nuclear magnetic resonance (NMR) spectroscopy. The crystal structure of the analogue [Pt(II)(mmap)(oxalate)] was determined using the single crystal X-ray diffraction method. Based upon a total of 4964 collected reflections, we determined that the compound crystallizes in the monoclinic space group P2(1)/c (with a=11.890(2) A, b=9.6695(19) A, c=9.875(2) A, beta=102.03(3) degrees, Z=4, and R=0.0428). In this complex, platinum has a slightly distorted square planar geometry with the two adjacent corners being occupied by two nitrogen atoms of the mmap ligand, whereas the remaining cis positions are occupied by two oxygen atoms of the oxalate molecule. The mmap ligand is in a boat conformation and forms six-membered chelating rings as well as the oxalate molecule forms five-membered chelating rings with platinum. The complexes were evaluated for their cytotoxic potential against the sensitive A2780 tumor model and cisplatin-resistant clone derived in vitro from potential cells.     

Structural variability and new intermolecular interactions of Z-DNA in crystals of d(pCpGpCpGpCpG).

We have determined the single crystal x-ray structure of the synthetic DNA hexamer d(pCpGpCpGpCpG) in two different crystal forms. The hexamer pCGCGCG has the Z-DNA conformation and in both cases the asymmetric unit contains more than one Z-DNA duplex. Crystals belong to the space group C222(1) with a = 69.73, b = 52.63, and c = 26.21 A, and to the space group P2(1) with a = 49.87, b = 41.26, c = 21.91 A, and gamma = 97.12 degrees. Both crystals show new crystal packing modes. The molecules also show striking new features when compared with previously determined Z-DNA structures: 1) the bases in one duplex have a large inclination with respect to the helical axis, which alters the overall shape of the molecule. 2) Some cytosine nitrogens interact by hydrogen bonding with phosphates in neighbor molecules. Similar base-phosphate interactions had been previously detected in some B-DNA crystals. 3) Basepair stacking between the ends of neighbor molecules is variable and no helical continuity is maintained between contiguous hexamer duplexes.     

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 of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.