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ABSTRACT Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the

membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped

pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3,4,5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that

is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of

the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active

ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from _Methanocaldococcus jannaschii_ or _Escherichia coli_ show the channel

in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active _E. coli_ ribosome–channel complex demonstrates that the nascent chain

opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the

SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STEPWISE GATING OF THE SEC61 PROTEIN-CONDUCTING CHANNEL BY SEC63 AND SEC62 Article 04 January 2021 VISUALIZATION OF

TRANSLATION AND PROTEIN BIOGENESIS AT THE ER MEMBRANE Article Open access 25 January 2023 RIBOSOME PROFILING REVEALS MULTIPLE ROLES OF SECA IN COTRANSLATIONAL PROTEIN EXPORT Article Open

access 13 June 2022 ACCESSION CODES PRIMARY ACCESSIONS ELECTRON MICROSCOPY DATA BANK * 5691 * 5692 * 5693 PROTEIN DATA BANK * 1VVK * 3J43 * 3J44 * 3J45 * 3J46 DATA DEPOSITS Electron density

maps have been submitted to the Electron Microscopy Data Bank (http://www.emdatabank.org/) under accession numbers EMD-5691, EMD-5692 and EMD-5693, and modelled structures to the Protein

Data Bank (http://www.rcsb.org/pdb/home/home.do) under accession numbers 3J43, 3J44, 3J45, 3J46 and 1VVK. REFERENCES * Park, E. & Rapoport, T. A. Mechanisms of Sec61/SecY-mediated

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CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank K. Matlack and T. Guettler for reading the manuscript. This work was supported by National Institutes of Health grants

GM067887 to J.C.G., GM080139 to S.J.L., GM052586 to T.A.R. and GM45377 to C.W.A. T.A.R. is a Howard Hughes Institute investigator. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of

Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA, Eunyong Park, Weikai Li, Andrew Whynot & Tom A. Rapoport

* Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, Massachusetts 02118-2526, USA, Jean-François Ménétret & Christopher W. Akey *

School of Physics, Georgia Institute of Technology, Atlanta, 30332, Georgia, USA James C. Gumbart * Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center

for Macromolecular Imaging, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA, Steven J. Ludtke Authors * Eunyong Park View author publications You can also search for

this author inPubMed Google Scholar * Jean-François Ménétret View author publications You can also search for this author inPubMed Google Scholar * James C. Gumbart View author publications

You can also search for this author inPubMed Google Scholar * Steven J. Ludtke View author publications You can also search for this author inPubMed Google Scholar * Weikai Li View author

publications You can also search for this author inPubMed Google Scholar * Andrew Whynot View author publications You can also search for this author inPubMed Google Scholar * Tom A.

Rapoport View author publications You can also search for this author inPubMed Google Scholar * Christopher W. Akey View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS E.P. designed and purified RNC–channel complexes, J.-F.M. and C.W.A. obtained and analysed the electron microscopy data, J.C.G. helped with MDFF and channel

modelling, S.J.L. helped with data analysis, W.L. and A.W. purified _M. jannaschii_ components, and T.A.R., E.P. and C.W.A. wrote the paper. CORRESPONDING AUTHORS Correspondence to Tom A.

Rapoport or Christopher W. Akey. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION This file

contains Supplementary Figures 1-22 and Supplementary Tables 1-3. (PDF 15975 kb) CONFORMATIONAL CHANGES IN THE E. COLI SECY CHANNEL DURING TRANSLOCATION - PART I The video illustrates

conformational changes in the SecY channel that occur when a nascent chain with a signal sequence is bound in a looped configuration. The nascent chain has been omitted for clarity. A morph

was generated between models of the closed and active SecY complex in Chimera, with the channel viewed from the front. The N-terminal half of SecY is in light blue, the C-terminal half is in

red, SecE is dark blue, SecG is brown, and the plug is yellow. Six isoleucines that line the pore are shown as ball and stick representations in dark grey. Ribosomal helices H6, H7, H50 and

H50 are shown in white; proteins L23, and L24 are green, and L29 is dark grey. The position and conformation of the 6/7 loop was fixed during the simulation. Note that SecE tilts to

accommodate movements of the two halves of SecY, and helix 8b moves downwards towards the lateral gate, while helix 9 remains anchored to the large ribosomal subunit. These movements open

the lateral gate between the two halves of SecY. (MOV 5307 kb) CONFORMATIONAL CHANGES IN THE E. COLI SECY CHANNEL DURING TRANSLOCATION - PART II The video illustrates conformational changes

in SecY channel that occur when a nascent chain with a signal sequence is bound in a looped configuration. The channel is viewed from the top. The nascent chain has been omitted for clarity.

Note that the N-terminal half of SecY rotates and tilts backwards, approximating a rigid body movement that also involves SecG. However, helix 3 bends and the junction between helix 5 and 6

also moves to accommodate these large movements. These movements create a large translocation pore with access to the opened lateral gate. (MOV 5618 kb) VISUALIZING THE NASCENT CHAIN IN THE

ACTIVE E. COLI SECY CHANNEL The video illustrates the orientation of a closed SecY channel beneath the ribosome and then shows a progression from a closed to an open SecY. The nascent chain

containing the signal sequence helix (residues 1-73; in green) is bound in a looped configuration and a cytoplasmic loop resides in a V-shaped canyon bounded by the 6/7 connection and helix

10. The N-terminal half of SecY is in light blue, the C-terminal half is in red, SecE is dark blue, SecG is brown, and the plug is yellow. Six isoleucines that line the pore are shown as

ball and stick representations in dark grey. Ribosomal helices H6, H7, H50 and H59 are shown in white; ribosomal proteins are colored as follows: L23 (purple), L29 (dark grey) and L29

(yellow). The extended nascent chain exiting the tunnel couldinteract with the 6/7 loop to help stabilize the translocating ribosomechannel complex. (MOV 12322 kb) POWERPOINT SLIDES

POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Park, E., Ménétret, JF., Gumbart, J. _et al._ Structure of the SecY channel during initiation of protein translocation. _Nature_ 506, 102–106 (2014).

https://doi.org/10.1038/nature12720 Download citation * Received: 11 June 2013 * Accepted: 04 October 2013 * Published: 23 October 2013 * Issue Date: 06 February 2014 * DOI:

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