Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure of the E. coli protein-conducting channel bound to a translating ribosome

Nature volume 438, pages 318–324 (2005)Cite this article

A Corrigendum to this article was published on 12 October 2006

Abstract

Secreted and membrane proteins are translocated across or into cell membranes through a protein-conducting channel (PCC). Here we present a cryo-electron microscopy reconstruction of the Escherichia coli PCC, SecYEG, complexed with the ribosome and a nascent chain containing a signal anchor. This reconstruction shows a messenger RNA, three transfer RNAs, the nascent chain, and detailed features of both a translocating PCC and a second, non-translocating PCC bound to mRNA hairpins. The translocating PCC forms connections with ribosomal RNA hairpins on two sides and ribosomal proteins at the back, leaving a frontal opening. Normal mode-based flexible fitting of the archaeal SecYEβ structure into the PCC electron microscopy densities favours a front-to-front arrangement of two SecYEG complexes in the PCC, and supports channel formation by the opening of two linked SecY halves during polypeptide translocation. On the basis of our observation in the translocating PCC of two segregated pores with different degrees of access to bulk lipid, we propose a model for co-translational protein translocation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: General features of the cryo-EM reconstruction of the E. coli RNC–SecYEG complex.
Figure 2: Normal mode-based flexible fitting (NMFF) of the SecYEG complex into cryo-EM density.
Figure 3: Stereo views of RNA and protein elements in the ribosome–PCC junction.
Figure 4: The path of the nascent chain through the ribosome and PCC.

Similar content being viewed by others

References

  1. Simon, S. M. & Blobel, G. A protein-conducting channel in the endoplasmic reticulum. Cell 65, 371–380 (1991)

    Article  CAS  Google Scholar 

  2. Wickner, W., Driessen, A. J. M. & Hartl, F. U. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu. Rev. Biochem. 60, 101–124 (1991)

    Article  CAS  Google Scholar 

  3. Brundage, L. et al. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell 62, 649–657 (1990)

    Article  CAS  Google Scholar 

  4. Gorlich, D. & Rapoport, T. A. Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane. Cell 75, 615–630 (1993)

    Article  CAS  Google Scholar 

  5. Gilmore, R. & Blobel, G. Translocation of secretory proteins across the microsomal membrane occurs through an environment accessible to aqueous perturbants. Cell 42, 497–505 (1985)

    Article  CAS  Google Scholar 

  6. Simon, S. M., Blobel, G. & Zimmerberg, J. Large aqueous channels in membrane vesicles derived from the rough endoplasmic reticulum of canine pancreas or the plasma membrane of Escherichia coli. Proc. Natl Acad. Sci. USA 86, 6176–6180 (1989)

    Article  ADS  CAS  Google Scholar 

  7. Mothes, W. et al. Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell 89, 523–533 (1997)

    Article  CAS  Google Scholar 

  8. Hessa, T. et al. Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433, 377–381 (2005)

    Article  ADS  CAS  Google Scholar 

  9. Beckmann, R. et al. Alignment of conduits for the nascent polypeptide chain in the ribosome–Sec61 complex. Science 278, 2123–2126 (1997)

    Article  ADS  CAS  Google Scholar 

  10. Menetret, J.-F. et al. The structure of ribosome–channel complexes engaged in protein translocation. Mol. Cell 6, 1219–1232 (2000)

    Article  CAS  Google Scholar 

  11. Beckmann, R. et al. Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107, 361–372 (2001)

    Article  CAS  Google Scholar 

  12. Morgan, D. G. et al. Structure of the mammalian ribosome-channel complex at 17 Å resolution. J. Mol. Biol. 324, 871–886 (2002)

    Article  CAS  Google Scholar 

  13. van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004)

    Article  CAS  Google Scholar 

  14. Rapoport, T. A., Goder, V., Heinrich, S. U. & Matlack, K. E. Membrane-protein integration and the role of the translocation channel. Trends Cell Biol. 14, 568–575 (2004)

    Article  CAS  Google Scholar 

  15. Prinz, A. et al. Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal tRNA. EMBO J. 19, 1900–1906 (2000)

    Article  CAS  Google Scholar 

  16. Raden, D., Song, W. & Gilmore, R. Role of the cytoplasmic segments of Sec61α in the ribosome-binding and translocation-promoting activities of the Sec61 complex. J. Cell Biol. 150, 53–64 (2000)

    Article  CAS  Google Scholar 

  17. Cheng, Z., Jiang, Y., Mandon, E. C. & Gilmore, R. Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation. J. Cell Biol. 168, 67–77 (2005)

    Article  CAS  Google Scholar 

  18. Nakatogawa, H. & Ito, K. The ribosomal exit tunnel functions as a discriminating gate. Cell 108, 629–636 (2002)

    Article  CAS  Google Scholar 

  19. Bessonneau, P., Besson, V., Collinson, I. & Duong, F. The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure. EMBO J. 21, 995–1003 (2002)

    Article  CAS  Google Scholar 

  20. van der Sluis, E. O., Nouwen, N. & Driessen, A. J. M. SecY–SecY and SecY–SecG contacts revealed by site-specific crosslinking. FEBS Lett. 527, 159–165 (2002)

    Article  CAS  Google Scholar 

  21. Tama, F., Miyashita, O. & Brooks, C. L. III NMFF: Flexible high-resolution annotation of low-resolution experimental data from cryo-EM maps using normal mode analysis. J. Struct. Biol. 147, 315–326 (2004)

    Article  CAS  Google Scholar 

  22. Go, N., Noguti, T. & Nishikawa, T. Dynamics of a small globular protein in terms of low-frequency vibrational modes. Proc. Natl Acad. Sci. USA 80, 3696–3700 (1983)

    Article  ADS  CAS  Google Scholar 

  23. Kaufmann, A. et al. Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighbouring SecE. Biochemistry 38, 9115–9125 (1999)

    Article  CAS  Google Scholar 

  24. Veenendaal, A., van der Does, C. & Driessen, A. Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis. J. Biol. Chem. 276, 32559–32566 (2001)

    Article  CAS  Google Scholar 

  25. Tani, K., Tokuda, H. & Mizushima, S. Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization. J. Biol. Chem. 265, 17341–17347 (1990)

    CAS  PubMed  Google Scholar 

  26. Wirth, A. et al. The Sec61p complex is a dynamic precursor activated channel. Mol. Cell 12, 261–268 (2003)

    Article  CAS  Google Scholar 

  27. Martoglio, B., Hofmann, M. W., Brunner, J. & Dobberstein, B. The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer. Cell 81, 207–214 (1995)

    Article  CAS  Google Scholar 

  28. Levy, R., Wiedmann, M. & Kreibich, G. In vitro binding of ribosomes to the β subunit of the Sec61p protein translocation complex. J. Biol. Chem. 276, 2340–2346 (2001)

    Article  CAS  Google Scholar 

  29. Nishiyama, K.-i., Mizushima, S. & Tokuda, H. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J. 12, 3409–3415 (1993)

    Article  CAS  Google Scholar 

  30. Schatz, P. J. et al. One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery. EMBO J. 10, 1749–1757 (1991)

    Article  CAS  Google Scholar 

  31. Kalies, K. U., Rapoport, T. A. & Hartmann, E. The β subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation. J. Cell Biol. 141, 887–894 (1998)

    Article  CAS  Google Scholar 

  32. Plath, K. et al. Signal sequence recognition in posttranslational protein transport across the yeast ER membrane. Cell 94, 795–807 (1998)

    Article  CAS  Google Scholar 

  33. Laird, V. & High, S. Discrete cross-linking products identified during membrane protein biosynthesis. J. Biol. Chem. 272, 1983–1989 (1997)

    Article  CAS  Google Scholar 

  34. Scotti, P. A. et al. YidC, the E. coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase. EMBO J. 19, 542–549 (2000)

    Article  CAS  Google Scholar 

  35. High, S. et al. Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence. J. Biol. Chem. 268, 26745–26751 (1993)

    CAS  PubMed  Google Scholar 

  36. Valent, Q. A. et al. The Escherichia coli SRP and SecB targeting pathways converge at the translocon. EMBO J. 17, 2504–2512 (1998)

    Article  CAS  Google Scholar 

  37. Neumann-Haefelin, C., Schafer, U., Muller, M. & Koch, H. G. SRP-dependent cotranslational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein. EMBO J. 19, 6419–6426 (2000)

    Article  CAS  Google Scholar 

  38. Zito, C. R. & Oliver, D. Two-stage binding of SecA to the bacterial translocon regulates ribosome–translocon interaction. J. Biol. Chem. 278, 40640–40646 (2003)

    Article  CAS  Google Scholar 

  39. Wagenknecht, T., Grassucci, R. & Frank, J. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli. J. Mol. Biol. 199, 137–147 (1988)

    Article  CAS  Google Scholar 

  40. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996)

    Article  CAS  Google Scholar 

  41. Valle, M. et al. Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process. EMBO J. 21, 3557–3567 (2002)

    Article  CAS  Google Scholar 

  42. Gabashvili, I. S. et al. Solution structure of the E. coli 70S ribosome at 11.5 Å resolution. Cell 100, 537–549 (2000)

    Article  CAS  Google Scholar 

  43. Chapman, M. S. Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron density function. Acta Crystallogr. A 51, 69–80 (1995)

    Article  Google Scholar 

  44. Tronrud, D. E., Ten Eyck, L. F. & Matthews, B. W. An efficient general-purpose least-squares refinement program for macromolecular structures. Acta Crystallogr. A 43, 489–501 (1987)

    Article  Google Scholar 

  45. Gao, H. et al. Study of the structural dynamics of the E. coli 70S ribosome using real-space refinement. Cell 113, 789–801 (2003)

    Article  CAS  Google Scholar 

  46. Harms, J. M. et al. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin. BMC Biol. 2, 4 (2004)

    Article  Google Scholar 

  47. Tirion, M. M. Large amplitude elastic motions in proteins from a single-parameter, atomic analysis. Phys. Rev. Lett. 77, 1905–1908 (1996)

    Article  ADS  CAS  Google Scholar 

  48. Breyton, C. et al. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 418, 662–665 (2002)

    Article  ADS  CAS  Google Scholar 

  49. Brunger, A. T. et al. Crystallography NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. A. Grassucci for training on the Tecnai F30 electron microscope; G. S. Allen for assistance with supervised classification, the software package O, and discussion of the manuscript; and M. Watters for assistance with the illustrations. N.B. thanks I. Berger and T. Ishikawa for advice on sample preparation and cryo-EM density symmetry analysis. This work was supported by Howard Hughes Medical Institute, National Science Foundation and National Institutes of Health (NIH) grants to J.F., and a Multiscale Modeling Tools for Structural Biology grant, funded by the NIH, to C.L.B. III. N.B. was supported by a grant from the Swiss National Science Foundation (SNSF), the NCCR Structural Biology program of the SNSF and a Young Investigator grant from the Human Frontier Science Program. C.S. was supported by post-doctoral fellowships from the Roche Research Foundation and the Ernst Schering Research Foundation. Author Contributions Grid preparation, cryo-EM, data processing, and atomic model generation, fitting, refinement and interpretation were done by K.M. (in the laboratory of J.F.). E. coli SecYEG–RNC complex preparation and PCC electron microscopy density symmetry analysis was performed by C.S. and S.J. (in the laboratory of N.B.). T.S. (J.F.) assisted in cryo-EM data processing. F.T. (in the laboratory of C.L.B. III) performed the NMA and NMFF.

Author information

Author notes

  1. Kakoli Mitra and Christiane Schaffitzel: *These authors contributed equally to this work

Authors and Affiliations

  1. Howard Hughes Medical Institute, Health Research Inc. at the Wadsworth Center, Empire State Plaza, New York, 12201-0509, Albany, USA

    Kakoli Mitra & Joachim Frank

  2. Institute for Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Hönggerberg, CH-8093, Zürich, Switzerland

    Christiane Schaffitzel, Simon Jenni & Nenad Ban

  3. Wadsworth Center, Empire State Plaza, NY, 12201-0509, Albany, USA

    Tanvir Shaikh & Joachim Frank

  4. Department of Molecular Biology, The Scripps Research Institute, 10550 Torrey Pines Road, California, 92037, La Jolla, USA

    Florence Tama & Charles L. Brooks III

  5. Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, New York, 12201-0509, Albany, USA

    Joachim Frank

Authors
  1. Kakoli Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christiane Schaffitzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tanvir Shaikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Florence Tama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simon Jenni
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles L. Brooks III
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nenad Ban
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Joachim Frank
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joachim Frank.

Ethics declarations

Competing interests

Coordinates for the translocating and non-translocating PCC have been deposited in the RCSB Protein Data Bank, with the accession codes 2AKI and 2AKH, respectively. The cryo-EM map of the E. coli RNC–SecYEG complex has been deposited in the EBI Macromolecular Structure Database, with the accession code EMD-1143. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Generation of RNC-PCC complexes. (PDF 68 kb)

Supplementary Figure 2

Selfrotation analysis of the non-translocating PCC EM density using the program GLRF. (PDF 268 kb)

Supplementary Figure 3

Normal mode analysis of the plug-less SecY complex. (PDF 1224 kb)

Supplementary Figure 4

Normal mode-based flexible fitting (NMFF) of the plug-less SecY complex. (PDF 1046 kb)

Supplementary Figure 5

Effect of resolution on the cryo-EM density of the translocating PCC. (PDF 73 kb)

Supplementary Notes

This file contains the Supplementary Results, Supplementary Discussion, Supplementary Figure Legends, Supplementary Methods and additional references. (DOC 76 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mitra, K., Schaffitzel, C., Shaikh, T. et al. Structure of the E. coli protein-conducting channel bound to a translating ribosome. Nature 438, 318–324 (2005). https://doi.org/10.1038/nature04133

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04133

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing