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Yorodumi- EMDB-3070: Mammalian ribosome bound to the native Sec61 protein-conducting c... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3070 | |||||||||
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Title | Mammalian ribosome bound to the native Sec61 protein-conducting channel in the idle state | |||||||||
Map data | Subtomogram average of non-solubilized ribosome-Sec61 complexes in the idle state. | |||||||||
Sample |
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Keywords | Ribosome / Sec61 / Translocon / Endoplasmic Reticulum / Cryoelectron Tomography / Subtomogram Analysis | |||||||||
Biological species | Canis lupus familiaris (dog) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 9.4 Å | |||||||||
Authors | Pfeffer S / Burbaum L / Unverdorben P / Pech M / Chen Y / Zimmermann R / Beckmann R / Foerster F | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3070.map.gz | 6.9 MB | EMDB map data format | |
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Header (meta data) | emd-3070-v30.xml emd-3070.xml | 9.9 KB 9.9 KB | Display Display | EMDB header |
Images | EMD-3070.tif | 209.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3070 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3070 | HTTPS FTP |
-Validation report
Summary document | emd_3070_validation.pdf.gz | 258.1 KB | Display | EMDB validaton report |
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Full document | emd_3070_full_validation.pdf.gz | 257.2 KB | Display | |
Data in XML | emd_3070_validation.xml.gz | 6.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3070 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3070 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_3070.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Subtomogram average of non-solubilized ribosome-Sec61 complexes in the idle state. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.62 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Mammalian ribosome bound to the native protein translocon on cani...
Entire | Name: Mammalian ribosome bound to the native protein translocon on canine pancreatic ER vesicles |
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Components |
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-Supramolecule #1000: Mammalian ribosome bound to the native protein translocon on cani...
Supramolecule | Name: 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
Supramolecule | Name: Membrane-bound 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
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Source (natural) | Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas |
-Macromolecule #1: ER protein translocon
Macromolecule | Name: ER protein translocon / type: protein_or_peptide / ID: 1 / Recombinant expression: No |
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Source (natural) | Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas / Organelle: Endoplasmic Reticulum |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 7.6 / Details: 20mM Hepes, 50mM KCl; 2mM MgCl2 |
Grid | Details: Lacey carbon molybdenum grid |
Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV / Method: Blot 3 seconds before plunging. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: Gatan |
Date | Jun 18, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 30 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -20 ° / Tilt series - Axis1 - Max angle: 20 ° |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Tomogram 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 in two consecutive steps focusing (I) on the translocon and (II) on the ribosomal tRNA binding sites. 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 using a 'conventional' subtomogram alignment procedure. |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.4 Å / Resolution method: OTHER / Software - Name: PyTom, tom_toolbox, av3_toolbox / Number subtomograms used: 12528 |
CTF correction | Details: Each tilt image |