title: The 1.25 A resolution refinement of the cholera toxin B-pentamer: evidence of peptide backbone strain at the receptor-binding site.
abstract: Crystals of the 61 kDa complex of the cholera toxin B-pentamer with the ganglioside GM1 receptor pentasaccharide diffract to near-atomic resolution. We have refined the crystallographic model for this complex using anisotropic displacement parameters for all atoms to a conventional crystallographic residual R=0.129 for all observed Bragg reflections in the resolution range 22 A to 1.25 A. Remarkably few residues show evidence of discrete conformational disorder. A notable exception is a minority conformation found for the Cys9 side-chain, which implies that the Cys9-Cys86 disulfide linkage is incompletely formed. In all five crystallographically independent instances, the peptide backbone in the region of the receptor-binding site shows evidence of strain, including unusual bond lengths and angles, and a highly non-planar (omega=153.7(7) degrees) peptide group between residues Gln49 and Val50. The location of well-ordered water molecules at the protein surface is notable reproduced among the five crystallographically independent copies of the peptide chain, both at the receptor-binding site and elsewhere. The 5-fold non-crystallographic symmetry of this complex allows an evaluation of the accuracy, reproducibility, and derived error estimates from refinement of large structures at near-atomic resolution. We find that blocked-matrix treatment of parameter covariance underestimates the uncertainty of atomic positions in the final model by approximately 10% relative to estimates based either on full-matrix inversion or on the 5-fold non-crystallographic symmetry.
authors.affiliation: Department of Biological Structure, Biomolecular Structure Center, University of Washington, Seattle, WA, 98195-7742, USA. merritt@u.washington.edu
authors.forename: E A
authors.initials: EA
authors.lastname: Merritt
authors.forename: P
authors.initials: P
authors.lastname: Kuhn
authors.forename: S
authors.initials: S
authors.lastname: Sarfaty
authors.forename: J L
authors.initials: JL
authors.lastname: Erbe
authors.forename: R K
authors.initials: RK
authors.lastname: Holmes
authors.forename: W G
authors.initials: WG
authors.lastname: Hol
mesh_terms: D001665:Binding Sites; D002772:Cholera Toxin / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006859:Hydrogen; D008958:Models, Molecular; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012997:Solvents
chemical_list: D011956:Receptors, Cell Surface; D012997:Solvents; C023552:choleragen receptor; D005677:G(M1) Ganglioside; D006859:Hydrogen; D002772:Cholera Toxin