Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A2.

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 A, and diffracted to 1.75 A resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 A, and diffracted to 2.6 A resolution. The crotapotin crystal diffracted to 2.3 A resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Crystal structure of alpha-galactosidase from Trichoderma reesei and its complex with galactose: implications for catalytic mechanism.

The crystal structures of alpha-galactosidase from the mesophilic fungus Trichoderma reesei and its complex with the competitive inhibitor, beta-d-galactose, have been determined at 1.54 A and 2.0 A resolution, respectively. The alpha-galactosidase structure was solved by the quick cryo-soaking method using a single Cs derivative. The refined crystallographic model of the alpha-galactosidase consists of two domains, an N-terminal catalytic domain of the (beta/alpha)8 barrel topology and a C-terminal domain which is formed by an antiparallel beta-structure. The protein contains four N-glycosylation sites located in the catalytic domain. Some of the oligosaccharides were found to participate in inter-domain contacts. The galactose molecule binds to the active site pocket located in the center of the barrel of the catalytic domain. Analysis of the alpha-galactosidase- galactose complex reveals the residues of the active site and offers a structural basis for identification of the putative mechanism of the enzymatic reaction. The structure of the alpha-galactosidase closely resembles those of the glycoside hydrolase family 27. The conservation of two catalytic Asp residues, identified for this family, is consistent with a double-displacement reaction mechanism for the alpha-galactosidase. Modeling of possible substrates into the active site reveals specific hydrogen bonds and hydrophobic interactions that could explain peculiarities of the enzyme kinetics.

Crystallization and preliminary X-ray diffraction analysis of a novel trypsin inhibitor from seeds of Copaifera langsdorffii.

A novel trypsin inhibitor isolated from seeds of Copaifera langsdorffii was purified to homogeneity and crystallized. Crystals suitable for X-ray analysis were grown using the hanging-drop vapour-diffusion method at 291 K in sodium acetate buffer at pH values near 4.3 using PEG 4000 as precipitant. The crystals presented symmetry compatible with the space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 58.71, c = 93.75 A, and diffracted to 1.83 A resolution at the synchrotron source.

Crystallization and preliminary X-ray study of haem-binding protein from the bloodsucking insect Rhodnius prolixus.

Rhodnius haem-binding protein (RHBP) from the bloodsucking insect Rhodnius prolixus, a 15 kDa protein, has been crystallized using polyethylene glycol as a precipitant. X-ray diffraction data have been collected at a synchrotron source. The crystals belong to the space group P4(1(3))2(1)2, with unit-cell parameters a = b = 64.98, c = 210.68 A, and diffract beyond 2.6 A resolution.

Purification, crystallization and preliminary X-ray study of beta-xylosidase from Trichoderma reesei.

An extracellular multifunctional beta-xylosidase was purified from a culture of the fungus Trichoderma reesei. The active 95 +/- 5 kDa enzyme has been crystallized from sodium acetate buffer using PEG as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.75, b = 98.54, c = 227.25 A, and diffract beyond 2.7 A resolution. X-ray data were collected from frozen crystals on a synchrotron source.