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Yorodumi- EMDB-2797: single-particle cryo reconstruction of the Large Ribosomal subuni... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2797 | |||||||||
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Title | single-particle cryo reconstruction of the Large Ribosomal subunit-associated protein Quality Control (RQC) complex | |||||||||
Map data | reconstruction of RQC-delta-RING complex generated after enriching for nonribosomal density using a likelihood-based classification algorithm | |||||||||
Sample |
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Keywords | RING domain E3 ubiquitin ligase / translational surveillance / protein quality control / cryo-EM / listerin/Ltn1 / Tae2/Nemf | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 9.6 Å | |||||||||
Authors | Lyumkis D / Oliveira dos Passos D / Tahara EB / Webb K / Bennett EJ / Vinterbo S / Potter CS / Carragher B / Joazeiro CAP | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2014 Title: Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex. Authors: Dmitry Lyumkis / Dario Oliveira dos Passos / Erich B Tahara / Kristofor Webb / Eric J Bennett / Staal Vinterbo / Clinton S Potter / Bridget Carragher / Claudio A P Joazeiro / Abstract: All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), ...All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. | |||||||||
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_2797.map.gz | 56.6 MB | EMDB map data format | |
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Header (meta data) | emd-2797-v30.xml emd-2797.xml | 9.9 KB 9.9 KB | Display Display | EMDB header |
Images | EMD-2797.png | 98.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2797 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2797 | 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_2797.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of RQC-delta-RING complex generated after enriching for nonribosomal density using a likelihood-based classification algorithm | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.69 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 : Large 60S ribosomal subunit in complex with protein quality contr...
Entire | Name: Large 60S ribosomal subunit in complex with protein quality control components |
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Components |
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-Supramolecule #1000: Large 60S ribosomal subunit in complex with protein quality contr...
Supramolecule | Name: Large 60S ribosomal subunit in complex with protein quality control components type: sample / ID: 1000 / Number unique components: 1 |
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Molecular weight | Theoretical: 2.5 MDa |
-Supramolecule #1: large 60S ribosomal subunit-associated protein quality control complex
Supramolecule | Name: large 60S ribosomal subunit-associated protein quality control complex type: complex / ID: 1 / Name.synonym: RQC complex / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: LSU 60S |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741 / synonym: Baker's yeast |
Molecular weight | Theoretical: 2.5 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 6.8 Details: 50 mM Hepes-KOH, 100 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 2 mM DTT, 2x protease inhibitors, 0.1% Igepal CA-630 |
Grid | Details: freshly plasma-cleaned holey carbon C-flat grid (Protochips) that had been overlaid with 2 nm thin carbon film |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER Method: specimens were prepared for cryo-EM by applying 3 microliters of sample to a freshly plasma-cleaned holey carbon C-flat grid (Protochips) that had been overlaid with 2 nm thin carbon film, ...Method: specimens were prepared for cryo-EM by applying 3 microliters of sample to a freshly plasma-cleaned holey carbon C-flat grid (Protochips) that had been overlaid with 2 nm thin carbon film, allowing the sample to adsorb to the grid for 30 s, followed by blotting with filter paper and plunge freezing into liquid ethane using a manual cryoplunger in an ambient environment at 4 C. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 92307 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000 |
Sample stage | Specimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Average: 93 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected by monitoring power spectra of continuously acquired images in leginon |
Date | Feb 23, 2013 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 3037 / Average electron dose: 32 e/Å2 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: each particle |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: OTHER / Software - Name: Frealign / Number images used: 77962 |
Details | see detailed methods in paper |