title: 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 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.
authors.affiliation: 1] Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany [2] Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Demers
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Habenstein
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Antoine
authors.initials: A
authors.lastname: Loquet
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Suresh
authors.initials: S
authors.lastname: Kumar Vasa
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Giller
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: 1] Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany [2] Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), 13125 Berlin, Germany [3] Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
authors.forename: Adam
authors.initials: A
authors.lastname: Lange
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Sgourakis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001703:Biophysics; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D017348:Microscopy, Electron, Scanning Transmission; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012763:Shigella flexneri / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C413572:MxiH protein, Shigella