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- EMDB-2099: Structure of ER membrane associated ribosomes in situ -

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Basic information

Entry
Database: EMDB / ID: EMD-2099
TitleStructure of ER membrane associated ribosomes in situ
Map dataReconstruction of ER membrane associated ribosome from canine pancreatic microsomes
Sample
  • Sample: ER membrane associated ribosome
  • Complex: Membrane-bound 80S ribosome
Keywords80S ribosome / translocon / mammalian / ER membrane
Biological speciesCanis lupus familiaris (dog)
Methodsubtomogram averaging / cryo EM / Resolution: 31.0 Å
AuthorsPfeffer S / Brandt F / Hrabe T / Lang S / Eibauer M / Zimmermann R / Foerster F
CitationJournal: Structure / Year: 2012
Title: Structure and 3D arrangement of endoplasmic reticulum membrane-associated ribosomes.
Authors: Stefan Pfeffer / Florian Brandt / Thomas Hrabe / Sven Lang / Matthias Eibauer / Richard Zimmermann / Friedrich Förster /
Abstract: In eukaryotic cells, cotranslational protein translocation across the endoplasmic reticulum (ER) membrane requires an elaborate macromolecular machinery. While structural details of ribosomes bound ...In eukaryotic cells, cotranslational protein translocation across the endoplasmic reticulum (ER) membrane requires an elaborate macromolecular machinery. While structural details of ribosomes bound to purified and solubilized constituents of the translocon have been elucidated in recent years, little structural knowledge of ribosomes bound to the complete ER protein translocation machinery in a native membrane environment exists. Here, we used cryoelectron tomography to provide a three-dimensional reconstruction of 80S ribosomes attached to functional canine pancreatic ER microsomes in situ. In the resulting subtomogram average at 31 Å resolution, we observe direct contact of ribosomal expansion segment ES27L and the membrane and distinguish several membrane-embedded and lumenal complexes, including Sec61, the TRAP complex and another large complex protruding 90 Å into the lumen. Membrane-associated ribosomes adopt a preferred three-dimensional arrangement that is likely specific for ER-associated polyribosomes and may explain the high translation efficiency of ER-associated ribosomes compared to their cytosolic counterparts.
History
DepositionMay 14, 2012-
Header (metadata) releaseJul 25, 2012-
Map releaseJul 25, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00895
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.00895
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2099-pic...

Downloads & links

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Map

FileDownload / File: emd_2099.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of ER membrane associated ribosome from canine pancreatic microsomes
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.68 Å/pix.
x 128 pix.
= 599.04 Å		4.68 Å/pix.
x 128 pix.
= 599.04 Å		4.68 Å/pix.
x 128 pix.
= 599.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.68 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00895 / Movie #1: 0.00895
Minimum - Maximum-0.05852651 - 0.08826262
Average (Standard dev.)0.00050413 (±0.007813)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 599.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.684.684.68
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z599.040599.040599.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0590.0880.001

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Supplemental data

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Sample components

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Entire : ER membrane associated ribosome

EntireName: ER membrane associated ribosome
Components
  • Sample: ER membrane associated ribosome
  • Complex: Membrane-bound 80S ribosome

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Supramolecule #1000: ER membrane associated ribosome

SupramoleculeName: ER membrane associated ribosome / type: sample / ID: 1000 / Details: The sample is embedded into its native membrane / Oligomeric state: ER membrane associated ribosome / Number unique components: 1
Molecular weightTheoretical: 4.5 MDa

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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 / Organelle: Endoplasmic reticulum / Location in cell: Endoplasmic reticulum
Molecular weightTheoretical: 4.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4
Details: 5 mM MgCl2, 140 mM KCl, 10 mM Hepes pH 7.4, 1 mM DTT, protease inhibitor
GridDetails: lacy carbon
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Specialist opticsEnergy filter - Name: Gatan GIF 2002 / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 10.0 eV
DateJan 1, 2011
Image recordingNumber real images: 328 / Average electron dose: 50 e/Å2 / Bits/pixel: 12
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using PyTom and classified by constrained principal component analysis. Average number of projections used in the 3D reconstructions: 1000. Average number of class averages: 1.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 31.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PyTom
CTF correctionDetails: each particle

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