High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy.

Abstract

We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state NMR (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the Type-III Secretion System (T3SS) needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent dataset of 691 ssNMR constraints and STEM measurements. The hybrid model resolves the conformation of the non-conserved N-terminus, that occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.