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- EMDB-0086: Template-free detection and classification of microsomal membrane... -

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Basic information

Entry
Database: EMDB / ID: EMD-0086
TitleTemplate-free detection and classification of microsomal membrane bound complexes
Map dataRibosome - free fully assembled translocon complex
Sample
  • Organelle or cellular component: Canine pancreatic ER vesicles
Biological speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 14.0 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 27, 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

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0086.png

Downloads & links

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Map

FileDownload / File: emd_0086.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome - free fully assembled translocon complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 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: 0.0592
Minimum - Maximum-0.3976593 - 0.65031487
Average (Standard dev.)0.0023532605 (±0.035282042)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Ribosome - free fully assembled translocon complex - 1st half

Fileemd_0086_half_map_1.map
AnnotationRibosome - free fully assembled translocon complex - 1st half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Ribosome - free fully assembled translocon complex - 2nd half

Fileemd_0086_half_map_2.map
AnnotationRibosome - free fully assembled translocon complex - 2nd half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Canine pancreatic ER vesicles

EntireName: Canine pancreatic ER vesicles
Components
  • Organelle or cellular component: Canine pancreatic ER vesicles

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Supramolecule #1: Canine pancreatic ER vesicles

SupramoleculeName: Canine pancreatic ER vesicles / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Details: The same sample as the one used for EMD-3068 - 72
Source (natural)Organism: Canis lupus familiaris (dog) / Organ: pancreas / Organelle: rough 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

Concentration2.0 mg/mL
BufferpH: 7.6
Component:
ConcentrationNameFormula
20.0 mMHepes
50.0 mMKCl
2.0 mMMgCl_2

Details: The same as for EMD-3068 - 72
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Details: Blot 3 seconds before plunging..
DetailsThe same sample was used for EMD-3068 - 72

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsThe same as for EMD-3068 - 72
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 0.75 e/Å2 / Details: The same as for EMD-3068 - 72
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 / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 8600
ExtractionNumber tomograms: 55 / Number images used: 64000 / Reference model: None / Method: Template-free, automatic
Details: Biological density was traced based on discrete Morse theory (DisPerSE software) and topological persistence. Particles were picked on the lumenal side of microsomes based on geometric ...Details: Biological density was traced based on discrete Morse theory (DisPerSE software) and topological persistence. Particles were picked on the lumenal side of microsomes based on geometric constraints and classified using affinity propagation clustering.
CTF correctionSoftware - Name: PyTom
Final 3D classificationNumber classes: 3 / Avg.num./class: 4000 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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