1. Howard Hughes Medical Institute, Health Research Inc. at the Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509, USA
2. Institute for Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland
3. Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509, USA
4. Department of Molecular Biology, The Scripps Research Institute, 10550 Torrey Pines Road, La Jolla, California 92037, USA
5. Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, New York 12201-0509, USA
6. *These authors contributed equally to this work

Correspondence to: Joachim Frank^1,3,5 Correspondence and requests for materials should be addressed to J.F. (Email: Joachim@wadsworth.org). 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.

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 SecYE 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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