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- EMDB-3068: Mammalian ribosome bound to the native Sec61 protein-conducting c... -

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Basic information

Entry
Database: EMDB / ID: EMD-3068
TitleMammalian ribosome bound to the native Sec61 protein-conducting channel in the 'non-inserting' state ('conventional' alignment)
Map dataSubtomogram average of non-solubilized ribosome-Sec61 complexes in the 'non-inserting' state. Subtomogram alignment was carried out using 'conventional' alignment.
Sample
  • Sample: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
  • Complex: Membrane-bound 80S ribosome
  • Protein or peptide: ER protein translocon
KeywordsRibosome / Sec61 / Translocon / Endoplasmic Reticulum / Cryoelectron Tomography / Subtomogram Analysis
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 speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 9.0 Å
AuthorsPfeffer S / Burbaum L / Unverdorben P / Pech M / Chen Y / Zimmermann R / Beckmann R / Foerster F
CitationJournal: Nat Commun / Year: 2015
Title: Structure of the native Sec61 protein-conducting channel.
Authors: Stefan Pfeffer / Laura Burbaum / Pia Unverdorben / Markus Pech / Yuxiang Chen / Richard Zimmermann / Roland Beckmann / Friedrich Förster /
Abstract: In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, ...In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment.
History
DepositionJul 1, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseSep 30, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a6u
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a6u
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5a6u
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3068.tif

Downloads & links

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Map

FileDownload / File: emd_3068.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of non-solubilized ribosome-Sec61 complexes in the 'non-inserting' state. Subtomogram alignment was carried out using 'conventional' alignment.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 220 pix.
= 576.4 Å		2.62 Å/pix.
x 220 pix.
= 576.4 Å		2.62 Å/pix.
x 220 pix.
= 576.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-16.746192929999999 - 20.314157489999999
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 576.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z576.400576.400576.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-16.74620.3140.000

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Supplemental data

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Sample components

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Entire : Mammalian ribosome bound to the native protein translocon on cani...

EntireName: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
Components
  • Sample: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
  • Complex: Membrane-bound 80S ribosome
  • Protein or peptide: ER protein translocon

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Supramolecule #1000: Mammalian ribosome bound to the native protein translocon on cani...

SupramoleculeName: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Membrane-bound 80S ribosome

SupramoleculeName: Membrane-bound 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas

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Macromolecule #1: ER protein translocon

MacromoleculeName: ER protein translocon / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas / Organelle: Endoplasmic Reticulum

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.6 / Details: 20mM Hepes, 50mM KCl; 2mM MgCl2
GridDetails: Lacey carbon molybdenum grid
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Method: Blot 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: Gatan
DateJun 18, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 30 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -20 ° / Tilt series - Axis1 - Max angle: 20 °
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsTomogram reconstruction and template matching against a single particle cryo-EM reconstruction of the 80S ribosome were accomplished using PyTom. Subtomograms extracted from cross correlation peaks in the tomogram were classified using constrained principal component analysis focusing on the large ribosomal subunit and the ER membrane. For the retained coordinates, 1 x binned subtomograms were reconstructed individually from the weighted back-projections using the full tilt range, iteratively aligned and classified focusing on the translocon. For the retained coordinates, unbinned subtomograms were reconstructed individually from the weighted back-projections using only a reduced tilt range (-20 deg to +20 deg) and iteratively aligned using a 'conventional' subtomogram alignment procedure.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: PyTom, tom_toolbox, av3_toolbox / Number subtomograms used: 17600
CTF correctionDetails: Each tilt image

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Atomic model buiding 1

Initial modelPDB ID:

3j7q


Chain - #0 - Chain ID: 1 / Chain - #1 - Chain ID: 2 / Chain - #2 - Chain ID: 3
SoftwareName: MDFF
DetailsThe Sec61a N-terminal domain along with Sec61b was first fitted as a rigid body prior to flexible fitting.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: pseudo-energy
Output model

PDB-5a6u:
Native mammalian ribosome-bound Sec61 protein-conducting channel in the 'non-inserting' state

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