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.     

Trigonal crystals of porcine mitochondrial aspartate aminotransferase

Crystals suitable for X-ray analysis of porcine mitochondrial aspartate aminotransferase in the closed conformation were obtained after the apoenzyme was reconstituted with N-5'-phosphopyridoxyl-L-aspartate, an inhibitor in which the cofactor is covalently bound to the substrate. This results in a crystal form that has not been encountered previously in studies of aspartate aminotransferases. The crystals belong to the trigonal space group P3121 (or the enantiomeric P3221) with unit cell dimensions alpha = b = 202.0 A, c = 58.0 A, alpha = beta = 90 degrees, gamma = 120 degrees and contain one dimer in the asymmetric unit.     

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.     

Crystallization and preliminary x-ray analysis of anti-obesity peptide hormone oxyntomodulin

Oxyntomodulin is a proglucagon-derived gut hormone that reduces food intake and body weight, thus represents a potential therapy for obesity. We synthesized and crystallized oxyntomodulin. The crystal diffracts x-ray to 2.4 A resolution and belongs to space group P2(1)3 with unit-cell parameters a=b=c= 48.44 A, alpha=beta=gamma=90 degrees . Preliminary analysis indicates a trimer packing in one asymmetric unit.     

Crystallization and preliminary X-ray crystallographic analysis of Ace: a collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.     

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 of chicken egg white cystatin, a low molecular weight protein inhibitor of cysteine proteinases, and preliminary X-ray diffraction data

The shorter-chain form of chicken egg white cystatin has been crystallized in 1.6 M-phosphate buffer at pH 4.0 by vapour diffusion. The crystals are of trigonal space group P3121 (or P3221), have cell constants a = b = 47.9 A, c = 87.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and contain one molecule of 12,191 molecular weight per asymmetric unit. They diffract well to about 2.0 A resolution and are suitable for X-ray crystal structure analysis.     

Crystallization and preliminary X-ray diffraction analysis of <Emphasis Type="Italic">E. coli</Emphasis> arginyl-tRNA synthetase in complex form with a tRNA<Superscript>Arg</Superscript>

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.     

Preliminary crystallographic data and primary sequence for anti-peptide Fab' B13I2 and its complex with the C-helix peptide from myohemerythrin

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a = b = 142.5 A, c = 101.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and Z = 1. The native Fab' crystallizes in space group P212121 with unit cell dimensions a = 98.0 A, b = 151.7 A, c = 80.8 A, alpha = beta = gamma = 90 degrees, and Z = 2. Both crystal forms diffract to beyond 2.6 A resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.     

Two-dimensional crystallization of Ca-ATPase by detergent removal.

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.     

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.     

Supramol--a program for structure analysis of intercalates using molecular simulations: the structure of VOPO4*C6H4O2

A method of structure analysis of intercalates has been developed that uses a combination of molecular simulations with powder diffraction. The program Supramol for the determination of intercalated structures uses crystal energy minimization in conjunction with powder diffraction data.The program solves the multiple minima problem in molecular mechanics, generating initial models systematically and searching for the global energy minimum by comparing the experimental and calculated diffraction patterns. The program is compatible with the Cerius2 modeling environment.Two intercalated crystal structures solved by Supramol are presented in the present paper: vanadyl phosphate intercalated with p-benzoquinone and the high temperature phase of vanadyl phosphate intercalated with dioxane. The structure of vanadyl phosphate intercalated with p-benzochinone is tetragonal, space group I4/ m, the unit cell parameters a=6.21 A, b=6.21 A, c=20.18 A and the density is rho=2.30 g x cm(-3), Z=4. The crystal structure of vanadyl phosphate intercalated with dioxane (high temperature phase) is monoclinic, space group C2/ m, unit cell parameters are: a= b=8.94 A, c=8.22 A, alpha=gamma=90 degrees, beta=106.30 degrees, Z=4, density 2.248 g x cm(-3).     

Crystallization and preliminary X-ray studies of annexin IV (endonexin), a calcium-dependent phospholipid-binding protein.

Annexin IV (endonexin) has been purified from chicken liver and crystallized by the vapour diffusion method. Crystals which diffract to at least 2.2 A have been obtained. They belong to space group R3 and have unit cell dimensions of a = b = 99.4 A, c = 96.2 A, alpha = 90 degrees, beta = 90 degrees, gamma = 120 degrees. There is one molecule of 32,500 Da per asymmetric unit.     

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.     

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.     

Molecular and crystal structure of (1----3)-alpha-D-glucan triacetate.

A crystal and molecular structure for GTA I, the low temperature polymorph of (1----3)-alpha-D-glucan triacetate, is proposed on the basis of X-ray diffraction analysis of well-oriented films, combined with stereochemical model refinement. The unit cell is monoclinic with parameters a = 30.17 A, b = 17.42 A, c (fibre axis) = 12.11 A, and beta = 90 degrees C. The probable space group is P2(1) with b axis unique. Six molecular chains pass through the unit cell with alternating polarity and with three independent chains comprising the asymmetric unit. The chain axes are located in a hexagonal packing arrangement. The chain backbone conformation is a left-handed, three-fold helix, but all nine O(6) acetyl groups of the asymmetric unit are in non-equivalent rotational positions. The most probable structure is indicated by X-ray residuals R = 0.261 and R" = 0.283, based on 62 reflection intensities (41 observed and 21 unobserved).     

Crystallization and preliminary X-ray crystallographic analysis of PAS factor from Vibrio vulnificus

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a=56.1, b=74.4, c=80.0 A, a=b=g=90 degrees. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 A using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.     

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.     

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.