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

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.

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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.

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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.

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Correspondence to Joachim Frank.

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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)

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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

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