Crystallization and preliminary X-ray diffraction study of Lathyrus ochrus isolectin II complexed to the human lactotransferrin N2 fragment.

Isolectin II (LOL II) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of the N2 fragment (18,500 Da) isolated from human lactotransferrin, which contains an N-acetyllactosamine type biantennary glycan linked to Asn137. This is the first example of a legume lectin crystallized with an N-glycosylprotein. Crystals of the LOL II-N2 complex belong to the tetragonal space group (P4(1)2(1)2 or the enantiomorph) with cell dimensions: a = b = 63.5 A, c = 251.9 A. They diffract well up to at least 3.5 A resolution and more weakly up to 2.8 A resolution. Assuming one functional half-entity in the asymmetric unit, an alpha, beta monomer complexed to one N2 fragment (24,500 Da + 18,500 Da) would give a Vm of 2.95 A3/Da and a solvent content of approximately 58%. SDS/polyacrylamide gels of the dissolved crystals show the presence of both the LOL II and N2 fragment.     

X-ray crystal structure determination and refinement at 1.9 A resolution of isolectin I from the seeds of Lathyrus ochrus

Orthorhombic crystals of isolectin I (LOLI) from the seeds of Lathyrus ochrus were first obtained during the STS 29 space shuttle mission. Subsequently, isostructural crystals were also obtained in the laboratory. They belong to the space group P2(1)2(1)2, with cell dimensions a = 135.84 A, b = 63.12 A and c = 54.54 A with one functional entity, a dimer, in the asymmetric unit (Vm = 2.2 A3/Da). The three-dimensional structure of LOLI, which was solved by the molecular replacement method using a 3 A resolution model of pea lectin, has subsequently been refined by using crystallographic data between 8.0 A and 1.9 A resolution, coupled to molecular dynamics and energy minimization techniques. The conventional R-factor is 0.185 for Fo greater than 1 sigma(Fo). The final model includes 220 well-defined water molecules and has root-mean-square deviations from ideal bond distances and angles of 0.004 A and 3 degrees, respectively. Only slight conformation differences have been found between the two alpha beta monomers. The molecular structure of LOLI, the first to be determined from the genus Lathyrus, is very similar to those of concanavalin A, pea lectin and favin. Differences are confined to the loop regions and beta-chain termini. Comparison of equivalent C alpha atom positions between our final model and the pea lectin structure shows slight differences in the association of the two monomers, which are probably due to the different environments in the crystals. The root-mean-square deviation between C alpha atoms of LOLI and pea lectin is 0.40 A. The metal binding sites are very similar in pea lectin, concanavalin A and LOLI. The sugar-binding sites of LOLI are occupied by four well-ordered water molecules each. The cleavage site for one of the monomers is specially well defined in the final electron density map: the amino group of Glul (alpha) seems to form a salt bridge with the carboxylate group of the terminal Asn181 (beta). A detailed analysis of the difference in crystal packing contacts between pea lectin and LOLI shows that, as might be expected, several of the intermolecular interactions are mediated by residues that correspond to substitutions in the LOLI amino acid sequence.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.     

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.     

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2₁ and have cell constants of a = 66.3 Å, b = 151.8 Å, c = 83.7 Å, and β = 101.2°. While larger crystals are twinned, smaller crystals (0.4 × 0.3 × 0.05 mm³) are single.     

Crystallization and preliminary X-ray studies of pyridoxal kinase from sheep brain

Crystals of pyridoxal kinase from sheep brain have been grown from solutions of ammonium sulfate or Na+/K+ phosphate. The crystals are trigonal, space group P3(1)21 with axes a = b = 102.2 A and c = 58.5 A. The crystals are quite stable to x-rays and diffract at 2.2-A resolution. This macromolecule is a 80,000-kDa dimer and utilizes the 2-fold symmetry of the space group, resulting in one subunit/asymmetric unit.     

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.     

Crystallization and preliminary X-ray diffraction studies of antigen--antibody complexes

Monoclonal antibodies of predefined specificity have been purified and crystallized as single components or complexed with their specific antigens. The intersegmental flexibility of antibody molecules has imposed the strategy of attempting to crystallize their Fab fragments separately. Intrasegmental mobility in Fabs has rarely been an obstacle to their crystallization. The immune system, however, provides a large functional and structural diversity of antibody molecules suitable for crystallization and X-ray diffraction studies.     

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.     

Crystallization and preliminary X-ray crystallographic studies of phosphoglycerate kinase from Thermus caldophilus

We report the purification and crystallization of phosphoglycerate kinase from Thermus caldophilus (Tca). The enzyme crystallizes in the P2(1)2(1)2(1) space group (cell dimensions a = 65.1, b = 71.3, c = 80.2 A), with one molecule in the asymmetric unit. A complete set of diffraction data was collected from an orthorhombic crystal up to 1.8 A resolution.     

Crystallization and preliminary X-ray studies of recombinant murine interferon-beta

Recombinant murine interferon-beta produced in Escherichia coli was purified and crystallized in an orthorhombic space group C222(1) with a = 61.67 A, b = 55.62 A, and c = 92.16 A. The crystals with a slight tendency for orientational disorder around the c axis diffract at least up to 3.3-A resolution. The crystallizability and the fact that the crystallographic asymmetric unit contains only one molecule of murine interferon-beta strongly indicate that the present preparation (Matsuda, S., Utsumi, J., and Kawano, G. (1986) J. Interferon Res., in press) of recombinant murine interferon-beta is predominantly homogeneous with respect to chemical, tertiary, and quaternary structures.     

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions $\text{a = 118.6}\text{A}\text{?}\text{, b = 88.9}\text{A}\text{?}\text{, c = 165.9}\text{A}\text{?}\text{, β = 103.0 °}$ and one tetramer of 120,000 M_r per asymmetric unit. The crystals are suitable for high-resolution work.     

Crystallization and preliminary X-ray studies of hydroxylamine oxidoreductase from Nitrosomonas europaea.

Hydroxylamine oxidoreductase [EC 1.7.3.4] from Nitrosomonas europaea was crystallized by the microdialysis method using ammonium sulfate. Its space group is P63 with cell dimensions of a = b = 96.4 A and c = 266.2 A. Its molecular weight was determined to be 190,000-195,000 by the X-ray small angle scattering and ultracentrifugal methods.     

Crystallization and preliminary X-ray crystallographic studies of rusticyanin from Thiobacillus ferrooxidans.

Rusticyanin is a 16.5 kDa type I blue copper protein isolated from Thiobacillus ferrooxidans. This organism can grow on Fe2+ as its sole energy source. Rusticyanin is thought to be a principal component in the iron respiratory electron transport chain of T. ferrooxidans. As a component of the periplasmic space of an acidophilic bacterium, rusticyanin is remarkably stable at acidic pH. It is redox-active down to pH 0.2. Crystals of rusticyanin have been grown from solutions of PEG 8000 by the hanging-drop vapor diffusion method. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions a = 32.36 A, b = 60.37 A, c = 74.60 A. The crystals diffract to 2.0 A resolution and they are stable in the X-ray beam for at least two days.     

Superoxide dismutase from Bacillus stearothermophilus: Crystallization and preliminary X-ray diffraction studies

Superoxide dismutase from Bacillus stearothermophilus is a dimeric manganese-containing enzyme with a molecular weight of 40,000. It has been crystallised in the monoclinic space group P2₁ with unit cell dimensions of $\text{a = 50}\text{A}\text{?}\text{, b = 70}\text{A}\text{?}\text{, c = 69}\text{A}\text{?}$ and β = 111 ° 10′. The asymmetric unit appears to be the dimer.     

Crystallization and preliminary X-ray diffraction studies of the cartilage link protein from bovine trachea

Cartilage extracellular matrix link protein, having molecular mass of approximately 40 kDa, is a metalloprotein that binds divalent cations and is only soluble in low ionic strength solutions. The link protein was purified from bovine trachea and has been crystallized by a vapor diffusion method using PEG 3350 as precipitant. The crystal symmetry is P1, and the unit cell dimensions are a = 43.55, b = 53.11, c = 60.10 Å, α = 90.44, β = 106.21, γ = 101.51°. The V_M of 1.8 Å₃/Da is consistent with the presence of two molecules of the link protein in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 Å resolution. © 1995 Wiley-Liss, Inc.