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We describe a straightforward crystallogenesis protocol leading to the preparation of protein crystals suitable for structure determination that involves protein expression and purification, refolding of the overexpressed protein, search of optimal crystallization conditions and diffraction data collection on native and selenomethionine substituted crystals. The protocol is exemplified with epididymal-specific lipocalin (rLcn6), a newly discovered monomeric protein of 19 kDa that may play an important role in sperm maturation. This protein was cloned from Norway rat (Rattus norvegicus) genome and expressed as insoluble inclusion bodies in Escherichia coli. After refolding of the purified protein, microcrystals were obtained after sparse matrix screening. Optimization of conditions after stepwise incremental initial conditions led to single crystals belonging to space group P212121 and data set at 1.90 A. After attempts with several models, initial phasing was not found by molecular replacement. A similar ...

Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB5 enterotoxin (choleragen). We have independently solved and refined the three-dimensional structure of choleragen at 2.5 A resolution. The structure of the crystalline toxin closely resembles that described for the heat-labile enterotoxin from Escherichia coli (LT) with which it shares 80% sequence homology. In both cases, the wedge-shaped A subunit is loosely held high above the plane of the pentameric B subunits by the tethering A2 chain. The most striking difference between the two toxins occurs at the carboxyl terminus of the A2 chain. Whereas the last 14 residues of the A2 chain of LT threading through the central pore of the B5 assembly form an extended chain with a terminal loop, the A2 chain of choleragen remains a nearly continuous alpha-helix throughout its length. The four carboxyl-terminal residues of the A2 chain (KDEL sequence), disordered in the crystal structure of LT, are clearly visible in choleragen's electron-density map. In the accompanying article we describe the three-dimensional structure of the isolated B pentamer of cholera toxin (choleragenoid). Comparison of the crystalline coordinates of choleragen, choleragenoid, and LT provides a solid three-dimensional foundation for further experimental investigation. These structures, along with those of related toxins from Shigella dysenteria and Bordetella pertussis, offer a first step towards the rational design of new vaccines and anti-microbial agents.