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- EMDB-0075: Template-free detection and classification of microsomal membrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-0075
TitleTemplate-free detection and classification of microsomal membrane bound complexes
Map dataRibosomal large subunit bound to fully assembled translocon complex
Sample
  • Complex: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles
Biological speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 21.0 Å
AuthorsMartinez-Sanchez A / Lucic V
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
DepositionJun 23, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseOct 2, 2019-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.318
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.318
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0075.png

Downloads & links

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Map

FileDownload / File: emd_0075.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosomal large subunit bound to fully assembled translocon complex
Voxel sizeX=Y=Z: 5.24 Å
Density
Contour LevelBy AUTHOR: 0.318 / Movie #1: 0.318
Minimum - Maximum-1.4439994 - 1.6727456
Average (Standard dev.)0.0059724436 (±0.11577764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 628.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.245.245.24
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z628.800628.800628.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-1.4441.6730.006

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Supplemental data

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Half map: Ribosomal large subunit bound to fully assembled translocon...

Fileemd_0075_half_map_1.map
AnnotationRibosomal large subunit bound to fully assembled translocon complex - 1st half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Ribosomal large subunit bound to fully assembled translocon...

Fileemd_0075_half_map_2.map
AnnotationRibosomal large subunit bound to fully assembled translocon complex - 2nd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
  • Complex: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles

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Supramolecule #1: 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: complex / ID: 1 / Parent: 0
Source (natural)Organism: Canis lupus familiaris (dog)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 7.6
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 55 / Number images used: 64000
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 873
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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