Crystallization and preliminary analysis of the deoxyoligonucleotide d(CGTAGATCTACG)

Two crystal forms of the self-complementary DNA 12-mer d(CGTAGATCTACG) were grown by the vapour diffusion technique. Form I is in space group C2 with a = 64.8 A, b = 35.4 A, c = 24.4 A and beta = 92.2 (1 A = 0.1 nm). The crystals are grown as monoclinic blocks or hexagonal plates. There are two strands (one duplex) in the asymmetric unit. Form II crystallizes as monoclinic blocks, space group P21 with a = 64.5 A, b = 35.1 A, c = 25.2 A and beta = 91.8 degrees. This form contains four strands (2 duplexes) in the asymmetric unit. Both forms are suitable for high resolution X-ray analysis. The diffraction patterns suggest that the DNA is in a B-type conformation and that the packing in the two forms is very similar.     

Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

Helix-forming tendencies of amino acids depend on the restrictions of side-chain rotamer conformations: crystal structure of the tripeptide GAI in two crystalline forms.

In our attempts to design crystalline alpha-helical peptides, we synthesized and crystallized GAI (C11H21N3O4) in two crystal forms, GAI1 and GAI2. Form 1 (GAI1) Gly-L-Ala-L-Ile (C11H21N3O4.3H2O) crystals are monoclinic, space group P2(1) with a = 8.171(2), b = 6.072(4), c = 16.443(4) A, beta = 101.24(2) degrees, V = 800 A3, Dc = 1.300 g cm-3 and Z = 2, R = 0.081 for 482 reflections. Form 2 (GAI2) Gly-L-Ala-L-Ile (C11H21N3O4.1/2H2O) is triclinic, space group P1 with a = 5.830(1), b = 8.832(2), c = 15.008(2) A, alpha = 102.88(1), beta = 101.16(2), gamma = 70.72(2) degrees, V = 705 A3, Z = 2, Dc = 1.264 g cm-3, R = 0.04 for 2582 reflections. GAI1 is isomorphous with GAV and forms a helix, whereas GAI2 does not. In GAI1, the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an incipient alpha-helix. GAI2 imitates a cyclic peptide and traps a water molecule. The conformation angles chi 11 and chi 12 for the side chain are (-63.7 degrees, 171.1 degrees) for the helical GAI1, and (-65.1 degrees, 58.6 degrees) and (-65.0 degrees, 58.9 degrees) for the two independent nonhelical molecules in GAI2; in GAI1, both the C gamma atoms point away from the helix, whereas in GAI2 the C gamma atom with the g+ conformation points inward to the helix and causes sterical interaction with atoms in the adjacent peptide plane. From these results, it is clear that the helix-forming tendencies of amino acids correlate with the restrictions of side-chain rotamer conformations. Both the peptide units in GAI1 are trans and show significant deviation from planarity [omega 1 = -168(1) degrees; omega 2 = -171(1) degrees] whereas both the peptide units in both the molecules A and B in GAI2 do not show significant deviation from planarity [omega 1 = 179.3(3) degrees; omega 2 = -179.3(3) degrees for molecule A and omega 1 = 179.5(3) degrees; omega 2 = -179.4(3) degrees for molecule B], indicating that the peptide planes in these incipient alpha-helical peptides are considerably bent.     

Crystallization and preliminary x-ray diffraction studies of a psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.     

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 the lactose-specific phosphocarrier protein IIAlac of the phosphoenolpyruvate: sugar phosphotransferase system from Staphylococcus aureus.

The IIA constituent of the lactose permease from Staphylococcus aureus has been crystallized in two different forms. Crystals of form I have been grown from polyethylene glycol 4000 with beta-octyl glucoside. They diffract to 3.0 A resolution and belong to space group C2 with unit cell dimensions a = 141.7 A, b = 130.7 A, c = 96.5 A and beta = 96.2 degrees. Form II crystals have been obtained from a solution containing polyethylene glycol 400, ammonium sulfate and manganese chloride. They diffract to at least 2.8 A resolution and belong to space group P2(1)2(1)2(1) with unit cell dimensions a = 89.9 A, b = 101.5 A and c = 90.9 A.     

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.     

Crystal structure of cholesteryl decanoate.

Cholesteryl decanoate (C37H64O2) is monoclinic, space group P2I, with cell dimensions a = 12.931 (6), b = 9.066 (2), c = 30.22 (1) A, beta = 91.14 (4) degrees, and Z = 4. The atomic coordinates from cholesteryl laurate were used in an initial trial structure which was refined by block diagonal least-squares methods with 1846 observed X-ray reflections (R = 0.129). Molecules A and B have almost fully extended conformations, except at the ester bonds and towards the end of the decanoate B chain. The molecules are arranged in antiparallel array forming monolayers of thickness d001 = 30.22 A, with the molecular long axis making an angle of about 67 degrees with the layer interface. The crystal structure is very similar to that of cholesteryl nonanoate and cholesteryl laurate.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

X-ray studies on crystalline complexes involving amino acids and peptides. XIX. Effects of change in chirality in the complexes of succinic acid with DL- and L-arginine

Crystals of DL-arginine hemisuccinate dihydrate (I)(monoclinic; P2(1)/c; a = 5.292, b = 16.296. c = 15.203 A; beta = 92.89 degrees; Z = 4) and L-arginine hemisuccinate hemisuccinic acid monohydrate (II) (triclinic; P1; a = 5.099; b = 10.222, c = 14.626 A; alpha = 77.31, beta = 89.46, gamma = 78.42 degrees; Z = 2) were grown under identical conditions from aqueous solutions of the components in molar proportions. The structures were solved by direct methods and refined to R = 0.068 for 2585 observed reflections in the case of (I) and R = 0.036 for 2154 observed reflections in the case of (II). Two of the three crystallographically independent arginine molecules in the complexes have conformations different from those observed so far in the crystal structures containing arginine. The succinic acid molecules and the succinate ions in the structures are centrosymmetric and planar. The crystal structure of (II) is highly pseudosymmetric. Arginine-succinate interactions in both the complexes involve specific guanidyl-carboxylate interactions. The basic elements of aggregation in both the structures are ribbons made up of alternating arginine dimers and succinate ions. However, the ribbons pack in different ways in the two structures. (II) presents an interesting case in which two ionisation states of the same molecule coexist in a crystal. The two complexes provide a good example of the effect of change in chirality on stoichiometry, conformation, aggregation, and ionisation state in the solid state.     

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.     

Synthetic peptide helices in crystals: structure and antiparallel and skewed packing motifs for alpha-helices in two isomeric decapeptides

The isomeric decapeptides Boc-Aib-Ala-Leu-Ala-Aib-Aib-Leu-Ala-Leu-Aib-OMe (II) and Boc-Aib-Ala-Aib-Ala-Leu-Ala-Leu-Aib-Leu-Aib-OMe (III), are predominantly alpha-helical with little effect on the conformation with interchange of Aib/Ala residues or Aib/Leu residues. The packing motif of helices in crystal II is antiparallel, whereas the helices pack in a skewed fashion in crystal III, with a 40 degrees angle between neighboring helix axes. Crystal III contains a water molecule in a hydrophobic hole that forms hydrogen bonds with two carbonyl oxygens that also participate in 5----1 type hydrogen bonds. Values for helical torsional angles phi and psi assume a much wider range than anticipated. Crystal II: C49H88N10O13, space group P2(1), with a = 16.625 (2) A, b = 9.811 (5) A, c = 18.412 (3) A, beta = 99.79 (1) degrees, Z = 2, R = 5.7% for 4338 data with magnitude of F0' greater than 3 sigma(F). Crystal III: C49H88N10O13 x 1/2H2O, space group P2(1) with a = 11.072 (2) A, b = 34.663 (5) A, c = 16.446 (3) A, beta = 107.85 (1) degrees, Z = 4, R = 8.3% for 6087 data with [F0[ greater than 3 sigma(F).     

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.     

Parallel and antiparallel aggregation of alpha-helices. Crystal structures of two apolar decapeptides X-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe (X = Boc, Ac)

An apolar helical decapeptide with different end groups, Boc- or Ac-, crystallizes in a completely parallel fashion for the Boc-analog and in an antiparallel fashion for the Ac-analog. In both crystals, the packing motif consists of rows of parallel molecules. In the Boc-crystals, adjacent rows assemble with the helix axes pointed in the same direction. In the Ac-crystals, adjacent rows assemble with the helix axes pointed in opposite directions. The conformations of the molecules in both crystals are quite similar, predominantly alpha-helical, except for the tryptophanyl side chain where chi 1 congruent to 60 degrees in the Boc- analog and congruent to 180 degrees in the Ac-analog. As a result, there is one lateral hydrogen bond between helices, N(1 epsilon)...O(7), in the Ac-analog. The structures do not provide a ready rationalization of packing preference in terms of side-chain interactions and do not support a major role for helix dipole interactions in determining helix orientation in crystals. The crystal parameters are as follow. Boc-analog: C60H97N11O13.C3H7OH, space group Pl with a = 10.250(3) A, b = 12.451(4) A, c = 15.077(6) A, alpha = 96.55(3) degrees, beta = 92.31(3) degrees, gamma = 106.37(3) degrees, Z = 1, R = 5.5% for 5581 data ([F] greater than 3.0 sigma(F)), resolution 0.89 A. Ac-analog: C57H91N11O12, space group P2(1) with a = 9.965(1) A, b = 19.707(3) A, c = 16.648(3) A, beta = 94.08(1), Z = 2, R = 7.2% for 2530 data ([F] greater than 3.0 sigma(F)), resolution 1.00 A.     

Thiourea derivatives and their nickel(II) and platinum(II) complexes: antifungal activity

We have synthesized two thiourea derivatives of methyl anthranilate (1, 2) and their complexes with nickel (3) and platinum(II) (4). We have also prepared the complexes of nickel(II) with two benzoylthiourea derivatives (5, 6). The obtained compounds were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-vis, NMR), mass spectrometry and thermal analysis. Compound 1, C(20)H(23)N(3)O(2)S, crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=8.8042(4) A, b=7.6608(3) A, c=28.834(2) A, alpha=gamma=90 degrees, beta=90.94(1) degrees. Compound 2, C(20)H(21)N(3)O(3)S, crystallizes in monoclinic space group P21/c, with Z=4, and unit cell parameters, a=7.7345(4) A, b=8.6715(4) A, c=29.113(2) A, alpha=gamma=90 degrees, beta=90.67(1) degrees. Compound 5, C(24)H(30)N(4)NiO(2)S(2), crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=10.4317(8) A, b=18.517(2) A, c=13.299(1) A, alpha=gamma=90 degrees, beta=104.53(1) degrees. Compound 6, C(25)H(28)Cl(2)N(4)NiO(4)S(2), crystallizes with a molecule of CH(2)Cl(2) in triclinic space group P-1, with Z=2, and unit cell parameters, a=10.362(1) A, b=11.849(2) A, c=12.536(2) A, alpha=90.04(2) degrees, beta=84.73(1) degrees, gamma=113.43(2) degrees. Compounds 1 and 2 show antifungal activity against the major pathogens responsible for important plant diseases (Botrytis cinerea, Colletotrichum fragariae, Fusarium oxysporum and Phoma betae). The antifungal activity is practically the same for morpholine and ethyl derivatives.     

Two crystal forms of helix II of Xenopus laevis 5S rRNA with a cytosine bulge

The crystal structure of r(GCCACCCUG).r(CAGGGUCGGC), helix II of the Xenopus laevis 5S rRNA with a cytosine bulge (underlined), has been determined in two forms at 2.2 A (Form I, space group P4(2)2(1)2, a = b = 57.15 A and c = 43.54 A) and 1.7 A (Form II, space group P4(3)2(1)2, a = b = 32.78 A and c = 102.5 A). The helical regions of the nonamers are found in the standard A-RNA conformations and the two forms have an RMS deviation of 0.75 A. However, the cytosine bulge adopts two significantly different conformations with an RMS deviation of 3.9 A. In Form I, the cytosine bulge forms an intermolecular C+*G.C triple in the major groove of a symmetry-related duplex with intermolecular hydrogen bonds between N4C and O6G, and between protonated N3+C and N7G. In contrast, a minor groove C*G.C triple is formed in Form II with intermolecular hydrogen bonds between O2C and N2G, and between N3C and N3G with a water bridge. A partial major groove opening was observed in Form I structure at the bulge site. Two Ca2+ ions were found in Form I helix whereas there were none in Form II. The structural comparison of these two forms indicates that bulged residues can adopt a variety of conformations with little perturbation to the global helix structure. This suggests that bulged residues could function as flexible latches in bridging double helical motifs and facilitate the folding of large RNA molecules.     

New crystal forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Three crystal forms of the dimeric form of the enzyme ribulose-1,5-bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum have been obtained from the gene product expressed in Escherichia coli. Form A crystals formed from the quaternary complex comprising enzyme-activator carbamate-Mg2+-2'-carboxyarabinitol-1,5-bisphosphate are shown here to be devoid of ligands. In contrast, crystals of the quaternary complex formed with the hexadecameric L8S8 enzyme from spinach contain both the activator carbamate and 2'-carboxyarabinitol-1,5-bisphosphate. Form B crystals of the R. rubrum enzyme are monoclinic, space group P2(1) with cell dimensions a = 65.5 A, b = 70.6 A, c = 104.1 A and beta = 92.1 degrees, with two subunits per asymmetric unit. Rotation function calculations show a non-crystallographic 2-fold axis perpendicular to the monoclinic b-axis. Form C crystals are orthorhombic (space group P2(1)2(1)2(1)) with cell dimensions a = 79.4 A, b = 100.1 A and c = 131.0 A. The monoclinic crystal form diffracts to at least 2.0 A resolution on a conventional X-ray source.     

Polymorphism of monolayer lipid membrane structures made from unsymmetrical bolaamphiphiles

The crystal structures of synthetic unsymmetrical 1-glucosamide- and 1-galactosamide bolaamphiphiles, 13-[(beta-d-glucopyranosyl)carbamoyl]tridecanoic acid (1) and 15-[(beta-d-galactopyranosyl)carbamoyl]pentadecanoic acid (2), respectively, were elucidated by single-crystal X-ray analysis. The space group for 1 is P2(1), Z=2 with cell dimensions: a=8.6816(9), b=4.8578(5), c=26.250(3)A, beta=91.460(2) degrees ; that for 2P2(1), Z=2 with cell dimensions: a=4.90(1), b=40.139(1), 6.289(1)A, beta=106.48(1) degrees . The glucopyranosyl and galactopyranosyl rings in 1 and 2, respectively, take a (4)C(1) chair conformation. In the crystal lattice, the 1-glucosamide 1 forms a symmetrical monolayer lipid membrane (MLM) structure in which the molecules are packed in an antiparallel fashion, while 1-galactosamide 2 has an unsymmetrical MLM with parallel molecular packing. The stereochemistry of the sugar hydroxy group proved to affect their hydrogen-bonding networks and induce the polymorphism of the MLM.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

Crystals of modified bovine neurophysin II

An enzymatically modified form of bovine neurophysin II has been crystallized in three unique crystal forms. The orthorhombic form crystallizes in space group P2(1)2(1)2 with a = 15.33 nm, b = 6.92 nm, c = 3.63 nm, with four molecules in the asymmetric unit. The monoclinic form crystallizes in space group P2(1) with a = 6.22 nm, b = 9.55 nm, c = 5.45 nm and beta = 110.2 degrees, with eight molecules in the asymmetric unit. The tetragonal form crystallizes in space group P4(1)2(1)2 or P4(3)2(1)2 with a = 14.1 nm and c = 14.2 nm, with twelve molecules in the asymmetric unit. We report here the crystallization conditions, as well as the crystal data.