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

Nat Commun. 2014 Sep 29;5:4976. doi: 10.1038/ncomms5976.

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 nuclear magnetic resonance (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 needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Biophysics
  • Cryoelectron Microscopy*
  • Crystallography, X-Ray
  • Escherichia coli / metabolism
  • Magnetic Resonance Spectroscopy*
  • Microscopy, Electron, Scanning Transmission
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Transport
  • Shigella flexneri / chemistry*

Substances

  • Bacterial Proteins
  • MxiH protein, Shigella

Associated data

  • PDB/2MME