THE 1.25 ANGSTROM RESOLUTION REFINEMENT OF THE CHOLERA-TOXIN B-PENTAMER - EVIDENCE OF PEPTIDE BACKBONE STRAIN AT THE RECEPTOR-BINDING SITE

Crystals of the 61 kDa complex of the cholera toxin B-pentamer with th e ganglioside G(M1) receptor pentasaccharide diffract to near-atomic r esolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventi onal crystallographic residual R = 0.129 for all observed Bragg reflec tions in the resolution range 22 Angstrom to 1.25 Angstrom. Remarkably few residues show evidence of discrete conformational disorder. A not able exception is a minority conformation found for the Cys9 side-chai n, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed, in all five crystallographically independent instances, the p eptide backbone in the region of the receptor-binding site shows evide nce of strain, including unusual bond lengths and angles, and a highly non-planar to = 153.7(7)degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protei n surface is notable reproduced among the five crystallographically in dependent copies of the peptide chain, both at the receptor-binding si te and elsewhere. The 5-fold noncrystallographic symmetry of this comp lex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic re solution. We find that blocked-matrix treatment of parameter covarianc e underestimates the uncertainty of atomic positions in the final mode l by approximately 10% relative to estimates based either on full-matr ix inversion or on the 5-fold non-crystallographic symmetry. (C) 1998 Academic Press.