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- EMDB-2512: Cryo-EM of the Sec61-complex bound to an 80S-RNC engaged with an ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2512
TitleCryo-EM of the Sec61-complex bound to an 80S-RNC engaged with an membrane-inserting LepM chain
Map dataReconstruction of Sec61 bound to a ribosome translating am inserting polypeptide (LepM)
Sample
  • Sample: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the membrane-inserting LepM-polypeptide
  • Complex: 80S Ribosome
  • Protein or peptide: Sec61-complex
KeywordsCo-translational protein translocation
Function / homology
Function and homology information


Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane ...Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / post-translational protein targeting to membrane, translocation / protein transmembrane transporter activity / guanyl-nucleotide exchange factor activity / phospholipid binding / ribosome binding / endoplasmic reticulum membrane / endoplasmic reticulum
Similarity search - Function
Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. ...Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY
Similarity search - Domain/homology
Protein transport protein Sec61 subunit alpha isoform 1 / Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta
Similarity search - Component
Biological speciesTriticum aestivum (bread wheat) / Canis lupus familiaris (dog)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsGogala M / Becker T / Beatrix B / Barrio-Garcia C / Berninghausen O / Beckmann R
CitationJournal: Nature / Year: 2014
Title: Structures of the Sec61 complex engaged in nascent peptide translocation or membrane insertion.
Authors: Marko Gogala / Thomas Becker / Birgitta Beatrix / Jean-Paul Armache / Clara Barrio-Garcia / Otto Berninghausen / Roland Beckmann /
Abstract: The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In ...The biogenesis of secretory as well as transmembrane proteins requires the activity of the universally conserved protein-conducting channel (PCC), the Sec61 complex (SecY complex in bacteria). In eukaryotic cells the PCC is located in the membrane of the endoplasmic reticulum where it can bind to translating ribosomes for co-translational protein transport. The Sec complex consists of three subunits (Sec61α, β and γ) and provides an aqueous environment for the translocation of hydrophilic peptides as well as a lateral opening in the Sec61α subunit that has been proposed to act as a gate for the membrane partitioning of hydrophobic domains. A plug helix and a so-called pore ring are believed to seal the PCC against ion flow and are proposed to rearrange for accommodation of translocating peptides. Several crystal and cryo-electron microscopy structures revealed different conformations of closed and partially open Sec61 and SecY complexes. However, in none of these samples has the translocation state been unambiguously defined biochemically. Here we present cryo-electron microscopy structures of ribosome-bound Sec61 complexes engaged in translocation or membrane insertion of nascent peptides. Our data show that a hydrophilic peptide can translocate through the Sec complex with an essentially closed lateral gate and an only slightly rearranged central channel. Membrane insertion of a hydrophobic domain seems to occur with the Sec complex opening the proposed lateral gate while rearranging the plug to maintain an ion permeability barrier. Taken together, we provide a structural model for the basic activities of the Sec61 complex as a protein-conducting channel.
History
DepositionNov 19, 2013-
Header (metadata) releaseNov 27, 2013-
Map releaseFeb 5, 2014-
UpdateSep 10, 2014-
Current statusSep 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cg6
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4cg6
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2512-Lep...

Downloads & links

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Map

FileDownload / File: emd_2512.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Sec61 bound to a ribosome translating am inserting polypeptide (LepM)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.24 Å/pix.
x 368 pix.
= 455.363 Å		1.24 Å/pix.
x 368 pix.
= 455.363 Å		1.24 Å/pix.
x 368 pix.
= 455.363 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2374 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.35129607 - 0.7926681
Average (Standard dev.)0.00147863 (±0.05871441)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 455.36322 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23739945652171.23739945652171.2373994565217
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z455.363455.363455.363
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S213
start NC/NR/NS-184-184-183
NC/NR/NS368368368
D min/max/mean-0.3510.7930.001

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Supplemental data

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Sample components

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Entire : Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating ...

EntireName: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the membrane-inserting LepM-polypeptide
Components
  • Sample: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the membrane-inserting LepM-polypeptide
  • Complex: 80S Ribosome
  • Protein or peptide: Sec61-complex

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Supramolecule #1000: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating ...

SupramoleculeName: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the membrane-inserting LepM-polypeptide
type: sample / ID: 1000 / Oligomeric state: One Sec61 binds to one 80S ribosome / Number unique components: 2

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Supramolecule #1: 80S Ribosome

SupramoleculeName: 80S Ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Triticum aestivum (bread wheat) / synonym: wheat germ

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Macromolecule #1: Sec61-complex

MacromoleculeName: Sec61-complex / type: protein_or_peptide / ID: 1 / Name.synonym: Dog pancreas Sec61-complex / Details: Data-subset resulted from computational sorting / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog / Cell: Pancreas Cell / Organelle: Endoplasmic Reticulum / Location in cell: ER-Membrane
Molecular weightTheoretical: 69.9 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Details: 30 mM Hepes/KOH 7.6, 10 mM Mg(OAc)2, 180 mM KOAC/HAc pH 7.6, 0.3 % Digitonin, 1 mM DTT
GridDetails: Quantifoil grids pre-coated with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJul 17, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 9805 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 148721 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.3 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using SIGNATURE, classified using MAPPOS and processed with SPIDER
CTF correctionDetails: On 3D-volume (SPIDER)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: OTHER / Software - Name: SPIDER, Signature, MAPPOS / Number images used: 30455

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Atomic model buiding 1

Initial modelPDB ID:

2wwb

SoftwareName: Chimera, Coot, MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4cg6:
Cryo-em of the Sec61-complex bound to the 80s ribosome translating a membrane-inserting substrate

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