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- EMDB-3069: Mammalian ribosome bound to the native Sec61 protein-conducting c... -

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Basic information

Entry
Database: EMDB / ID: EMD-3069
TitleMammalian ribosome bound to the native Sec61 protein-conducting channel in the 'non-inserting' state ('gold standard' alignment)
Map dataSubtomogram average of non-solubilized ribosome-Sec61 complexes in the 'non-inserting' state. Subtomogram alignment was carried out following the 'gold standard' procedure implemented in PyTom.
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
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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3069.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 following the 'gold standard' procedure implemented in PyTom.
Voxel sizeX=Y=Z: 2.62 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-16.74375534 - 20.823352809999999
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-16.74420.823-0.000

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

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Image processing

CTF correctionDetails: Each tilt image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PyTom, tom_toolbox, av3_toolbox / Number subtomograms used: 17600
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 following the 'gold standard' subtomogram alignment procedure.

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