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- EMDB-3072: Representative tomogram of canine pancreatic rough Endoplasmic Re... -

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Basic information

Entry
Database: EMDB / ID: EMD-3072
TitleRepresentative tomogram of canine pancreatic rough Endoplasmic Reticulum vesicles
Map dataRepresentative tomogram of canine pancreatic rough Endoplasmic Reticulum vesicles.
Sample
  • Sample: Representative tomogram of canine pancreatic rough Endoplasmic Reticulum 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)
Methodelectron tomography / cryo EM
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-
UpdateMar 2, 2016-
Current statusMar 2, 2016Processing site: PDBe / Status: Released

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Structure visualization

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  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

EMD-3072.tif

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Map

FileDownload / File: emd_3072.map.gz / Format: CCP4 / Size: 101.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRepresentative tomogram of canine pancreatic rough Endoplasmic Reticulum vesicles.
Voxel sizeX=Y=Z: 20.96 Å
Density
Minimum - Maximum-25.035238270000001 - 32.838691709999999
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions462462128
Spacing462462128
CellA: 9683.52 Å / B: 9683.52 Å / C: 2682.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z20.9620.9620.96
M x/y/z462462128
origin x/y/z0.0000.0000.000
length x/y/z9683.5209683.5202682.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS462462128
D min/max/mean-25.03532.8390.000

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Supplemental data

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Sample components

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Entire : Representative tomogram of canine pancreatic rough Endoplasmic Re...

EntireName: Representative tomogram of canine pancreatic rough Endoplasmic Reticulum vesicles.
Components
  • Sample: Representative tomogram of canine pancreatic rough Endoplasmic Reticulum vesicles.
  • Complex: Membrane-bound 80S ribosome
  • Protein or peptide: ER protein translocon

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Supramolecule #1000: Representative tomogram of canine pancreatic rough Endoplasmic Re...

SupramoleculeName: Representative tomogram of canine pancreatic rough Endoplasmic Reticulum 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 / Database: NCBI / 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 / Database: NCBI
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
Processingelectron tomography
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: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
DateJun 18, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 100 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each tilt image
Final reconstructionSoftware - Name: PyTom, tom_toolbox, av3_toolbox / Number images used: 61
DetailsTomogram reconstruction was accomplished using PyTom.

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