Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.     

Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants

The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized. Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies. The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A. These crystals diffract to at least 1.8 A resolution. The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees. These crystals diffract to at least 2.0 A resolution.     

Crystallization of an anti-2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl monoclonal antibody Fab fragment with and without bound hapten

The Fab fragment of a monoclonal antibody, AN02, specific for a 2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl hapten was crystallized both with and without bound hapten. Both crystals formed in phosphate-buffered saline (150 mM-NaCl, 10 mM-Na2PO4, 0.02% (w/v) NaN3 (pH 7.4) at 4 degrees C and diffracted beyond 2.2 A resolution (1A = 0.1 nm). Fab with bound hapten crystallizes in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 73.23 A, c = 373.8 A, alpha = beta = 90 degrees and gamma = 120 degrees. Unoccupied Fab also crystallizes in space group P6(1)22 or P6(5)22 with cell dimensions a = b = 73 A, c = 377 A, alpha = beta = 90 degrees and gamma = 120 degrees.     

Crystallization and preliminary X-ray diffraction studies of aspartic proteinase from Irpex lacteus.

Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.     

Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog

The Fab′ fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions a = 37.1 Å, b = 63.3 Å, c = 178.5 Å, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 Å and are suitable for X-ray crystallographic studies. © 1994 John Wiley & Sons, Inc.     

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.     

Biochemical implications from the variable gene sequences of an anti-cytochrome c antibody and crystallographic characterization of its antigen-binding fragment in free and antigen-complexed forms.

To study the nature of antibody-antigen interactions, we have determined the variable gene sequences of the anti-cytochrome c immunoglobulin G1 (IgG1) monoclonal antibody E8, and obtained diffraction-quality crystals of the E8 antigen-binding fragment (Fab), both free and bound to its antigen, horse cytochrome c. The FabE8 crystals belong to space group P21 with unit cell dimensions of a = 45.0 A, b = 85.1 A, c = 63.3 A and beta = 105.5 degrees, have one FabE8 molecule per asymmetric unit and diffract to at least 2.1 A resolution. Crystals of the FabE8-cytochrome c complex belong to space group P212121 with unit cell dimensions of a = 84.3 A, b = 73.3 A and c = 94.9 A, accommodate one complex per asymmetric unit and diffract to 2.4 A resolution. In the nucleotide-derived amino acid sequences, the light-chain variable domain (VL) but not the heavy-chain variable domain (VH) of E8 is nearly identical to that of the anti-lysozyme antibody D1.3, differing by only five amino acid residues. Only one of these interacts with lysozyme in the D1.3-lysozyme crystal structure. Six negative and four positive charges in the VH complementarity determining regions of E8 complement four positive and three negative charges in the E8 epitope on cytochrome c. These data suggest that only a subset of the residues in an antibody-protein interface may be critical for binding and that the VH may play a dominant role in antigenic recognition.     

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.     

Preliminary crystallographic studies of the Fab fragment of an anti-azophenylarsonate antibody

Single crystals of the Fab fragment of a murine A/J anti-azophenylarsonate monoclonal antibody have been prepared by the vapor diffusion method. Antibody 3A7 uses the same combination of variable region gene segments (VK, JK, VH, JH) as do anti-azophenylarsonate antibodies bearing a predominant cross-reactive idiotype, but utilizes a different D gene segment. The crystals grow in the presence of beta-octylglucoside as tetragonal bipyramids in the space group of either P4(1)2(1)2 or P4(3)3(1)2 and with unit cell dimensions of a = b = 77.9 A, and c = 146.7 A. They diffract X-rays to better than 2.7 A resolution. Data up to 2.7 A resolution have been collected.     

Crystallization,preliminary X-ray diffraction study,and crystal packing of a complex between anti-Hen lysozyme antibody F9.13.7 and Guinea-fowl lysozyme

The complex formed between the Fab fragment of a murine monoclonal anti-hen egg lysozyme antibody F9.13.7 and the het-erologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 Å resolution, belong to the monoclinic space group P2₁, with a = 83.7 Å, b = 195.5 Å, c = 50.2 Å, β = 108.5° and have two molecules of the complex in the asymmetric unit The three-dimensional structure has been determined from a preliminary data set to 4 Å using molecular replacement techniques. The lysozyme–Fab complexes are arranged with their long molecular axes approximately parallel to the crystallo-graphic unique axis. Fab F9.13.7 binds an anti-genie determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10. © 1993 Wiley-Liss, Inc.     

Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Preliminary crystallographic study of pyrimidine dimer-specific excision-repair enzyme from bacteriophage T4

Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2(1), with unit cell parameters: a = 41.4 A, b = 40.1 A, c = 37.5 A, beta = 90.01 degrees. The unit cell contains two 16,000 Mr molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.     

Crystallization of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain "pathogenesis-related" proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine's low pH optimum (pH 4.0), we have crystallized two hevamine isozymes as a first step towards a high-resolution X-ray structure determination. Suitable crystals were obtained at room temperature from hanging drop experiments by vapor diffusion against 1.7 M to 3.4 M-NaCl (pH 5.0 to 9.0) for the major isozyme, and by vapor diffusion against 2.5 M to 4.3 M-NaCl (pH 5.0 to 8.0) for the minor one. Both isozymes give the same crystal morphology and space group. Their space group is P2(1)2(1)2(1) with cell dimensions a = 82.3 A, b = 58.1 A and c = 52.5 A (1 A = 0.1 nm). The crystals diffract to at least 2.0 A resolution.     

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 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.     

Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies

The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V_H-V_L interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V_H-V_L interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.     

Preliminary crystallographic study of a complex between an heteroclitic anti-hen egg-white lysozyme antibody and the heterologous antigen pheasant egg-white lysozyme

We report the preparation, crystallization and preliminary X-ray crystallographic study of the Fab fragment from a heteroclitic murine (BALB/c) monoclonal anti-hen egg-white lysozyme antibody complexed with a heterologous antigen, pheasant lysozyme. The complex between the heterologous antigen and the antibody has been crystallized from polyethylene glycol 8000 solutions in a form suitable for X-ray crystallographic studies. The crystals are monoclinic, space group C2 with a = 158.2 A, b = 49.1 A, c = 177.6 A, beta = 92.0 degrees (1 A = 0.1 nm).     

A new crystal form of the complex between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus that diffracts to 2.8 A resolution.

Two distinct complexes between seryl-tRNA synthetase and tRNA(Ser) from Thermus thermophilus have been crystallized using ammonium sulphate as a precipitant. Form III crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA. They are of monoclinic space group C2 with unit cell dimensions a = 211.6 A, b = 126.8 A, c = 197.1 A, beta = 132.4 degrees and diffract to about 3.5 A. Preliminary crystallographic results show that the crystallographic asymmetric unit contains two synthetase dimers. Form IV crystals grow from solutions containing a 1:1.5 stoichiometry of synthetase dimer to tRNA. They are of orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 124.5 A, b = 128.9 A, c = 121.2 A and diffract to 2.8 A resolution. Preliminary crystallographic results show that these crystals contain only one tRNA molecule bound to a synthetase dimer.     

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.