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Cryo-EM structure of the ribosome–SecYE complex in the membrane environment

Nature Structural & Molecular Biology volume 18, pages 614–621 (2011)Cite this article

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Abstract

The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.

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Figure 1: Reconstitution and cryo-EM reconstruction of a 70S RNC–Nd–SecYEG complex.
Figure 2: Structure of the Nanodisc.
Figure 3: Structure and connections of the membrane-embedded open SecYEG–RNC complex.
Figure 4: Path of the nascent chain and signal anchor.
Figure 5: Molecular dynamics simulation and membrane insertion.

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Acknowledgements

We thank B. Seidelt for help with the RNC preparation, J.P. Armache for help with data processing, S. Feuerstein (Forschungszentrum Jülich) for providing the plasmid encoding the apolipoprotein construct, J. Buerger and C. Ungewickell for help with the electron microscopy and D. Wilson for critical discussions. This research was supported by a Boehringer Ingelheim Fonds fellowship (to J.F.), by an Alexander von Humboldt Foundation fellowship and a Human Frontiers Science Program long term fellowship (to E.O.v.d.S.), by a Dr. Klaus Römer Stiftung fellowship (to T.B.), by grants from the Deutsche Forschungsgemeinschaft SFB594 and SFB646 (to R.B.) and SFB 740 (to T.M.), by US National Institutes of Health grants P41-RR005969 and R01-GM067887 (to K.S.), by US National Science Foundation (NSF) grant PHY0822613 (to K.S.) by an Alexander von Humboldt Foundation award (to K.S.) and by the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork, Anwenderzentrum). Computer time for MD simulations and MDFF was provided through NSF Large Resources Allocation Committee grant MCA93S028.

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Authors and Affiliations

  1. Department for Biochemistry, Gene Center, University of Munich, Munich, Germany

    Jens Frauenfeld, Eli O van der Sluis, Marco Gartmann, Birgitta Beatrix, Otto Berninghausen, Thomas Becker & Roland Beckmann

  2. Department of Chemistry and Biochemistry, Munich Center for Integrated Protein Science (CIPSM), Munich, Germany

    Jens Frauenfeld, Eli O van der Sluis, Marco Gartmann, Birgitta Beatrix, Otto Berninghausen, Thomas Becker & Roland Beckmann

  3. Department of Physics, Beckman Institute, University of Illinois at Urbana–Champaign, Urbana, Illinois, USA

    James Gumbart & Klaus Schulten

  4. Departamento de Genética Molecular, Instituto de Fisiología Celular, Circuito Exterior S/N, Ciudad Universitaria, Universidad Nacional Autónoma de México, Mexico City, Mexico

    Soledad Funes

  5. Ultrastrukturnetzwerk, Max Planck Institute for Molecular Genetics, Institut für Medizinische Physik und Biophysik, Charite–Universitätsmedizin Berlin, Berlin, Germany

    Thorsten Mielke

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Contributions

J.F. prepared the sample, collected the EM data, performed the 3D reconstruction and built the molecular model; J.G. did the MDFF and the MD simulations. E.O.v.d.S., S.F. and B.B. contributed to the purification of SecYEG; M.G. contributed to the data processing. T.M. and O.B. contributed to the EM data collection. T.B. contributed to model building and interpretation. All authors contributed to the study design and to writing the manuscript.

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Correspondence to Roland Beckmann.

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Supplementary Methods, Supplementary Figures 1–12 and Supplementary Tables 1–7 (PDF 3367 kb)

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Molecular dynamics simulation (MOV 29075 kb)

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Frauenfeld, J., Gumbart, J., Sluis, E. et al. Cryo-EM structure of the ribosome–SecYE complex in the membrane environment. Nat Struct Mol Biol 18, 614–621 (2011). https://doi.org/10.1038/nsmb.2026

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