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Yorodumi- EMDB-2704: Cryo-EM structure of a mammalian 80S ribosome-nascent chain complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2704 | |||||||||
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Title | Cryo-EM structure of a mammalian 80S ribosome-nascent chain complex | |||||||||
Map data | stalled mammalian 80S ribosome-nascent chain complex | |||||||||
Sample |
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Keywords | ribosome / ubiquitination / quality control | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.8 Å | |||||||||
Authors | Shao S / Hegde RS | |||||||||
Citation | Journal: Mol Cell / Year: 2014 Title: Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors. Authors: Sichen Shao / Ramanujan S Hegde / Abstract: Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase ...Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway. | |||||||||
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_2704.map.gz | 17.8 MB | EMDB map data format | |
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Header (meta data) | emd-2704-v30.xml emd-2704.xml | 8.1 KB 8.1 KB | Display Display | EMDB header |
Images | emd_2704.tif | 173.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2704 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2704 | HTTPS FTP |
-Validation report
Summary document | emd_2704_validation.pdf.gz | 255.3 KB | Display | EMDB validaton report |
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Full document | emd_2704_full_validation.pdf.gz | 254.4 KB | Display | |
Data in XML | emd_2704_validation.xml.gz | 6.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2704 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2704 | 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_2704.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | stalled mammalian 80S ribosome-nascent chain complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.34 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 : stalled mammalian 80S ribosome-nascent chain complex
Entire | Name: stalled mammalian 80S ribosome-nascent chain complex |
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Components |
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-Supramolecule #1000: stalled mammalian 80S ribosome-nascent chain complex
Supramolecule | Name: stalled mammalian 80S ribosome-nascent chain complex / type: sample / ID: 1000 / Number unique components: 1 |
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-Supramolecule #1: 80S ribosome
Supramolecule | Name: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 / Details: 50 mM Hepes, 100 mM KAc, 5 mM MgAc2, 1 mM DTT |
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Grid | Details: Quantifoil R2/2 on 400 mesh Cu grid with thin carbon support |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: Blot 3 seconds before plunging |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification |
Date | Apr 1, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1568 / Average electron dose: 25 e/Å2 Details: An in-house system was used to intercept the videos from the detector at a rate of 17 frames for 1s exposures |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | RELION movement correction processing |
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CTF correction | Details: each particle |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 44255 |