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Yorodumi- EMDB-2832: Cryo-EM map of a mammalian 60S ribosome-nascent chain-tRNA comple... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2832 | |||||||||
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Title | Cryo-EM map of a mammalian 60S ribosome-nascent chain-tRNA complex with Listerin and NEMF | |||||||||
Map data | reconstruction of the rabbit 60S ribosomal subunit housing a defined nascent chain-P-site tRNA in complex with the E3 ligase Listerin and NEMF | |||||||||
Sample |
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Keywords | ribosome-associated quality control / ubiquitin ligase | |||||||||
Function / homology | Function and homology information alpha-aminoacyl-tRNA binding / CAT tailing / RQC complex / nuclear export / ribosome-associated ubiquitin-dependent protein catabolic process / ribosomal large subunit binding / protein autoubiquitination / rescue of stalled ribosome / cytosolic ribosome / RING-type E3 ubiquitin transferase ...alpha-aminoacyl-tRNA binding / CAT tailing / RQC complex / nuclear export / ribosome-associated ubiquitin-dependent protein catabolic process / ribosomal large subunit binding / protein autoubiquitination / rescue of stalled ribosome / cytosolic ribosome / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / protein-containing complex assembly / tRNA binding / zinc ion binding / nucleus / cytosol Similarity search - Function | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) / Sus scrofa (pig) / unidentified (others) / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
Authors | Shao S / Brown A / Santhanam B / Hegde RS | |||||||||
Citation | Journal: Mol Cell / Year: 2015 Title: Structure and assembly pathway of the ribosome quality control complex. Authors: Sichen Shao / Alan Brown / Balaji Santhanam / Ramanujan S Hegde / Abstract: During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ...During ribosome-associated quality control, stalled ribosomes are split into subunits and the 60S-housed nascent polypeptides are poly-ubiquitinated by Listerin. How this low-abundance ubiquitin ligase targets rare stall-generated 60S among numerous empty 60S is unknown. Here, we show that Listerin specificity for nascent chain-60S complexes depends on nuclear export mediator factor (NEMF). The 3.6 Å cryo-EM structure of a nascent chain-containing 60S-Listerin-NEMF complex revealed that NEMF makes multiple simultaneous contacts with 60S and peptidyl-tRNA to sense nascent chain occupancy. Structural and mutational analyses showed that ribosome-bound NEMF recruits and stabilizes Listerin's N-terminal domain, while Listerin's C-terminal RWD domain directly contacts the ribosome to position the adjacent ligase domain near the nascent polypeptide exit tunnel. Thus, highly specific nascent chain targeting by Listerin is imparted by the avidity gained from a multivalent network of context-specific individually weak interactions, highlighting a new principle of client recognition during protein quality control. | |||||||||
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_2832.map.gz | 15.6 MB | EMDB map data format | |
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Header (meta data) | emd-2832-v30.xml emd-2832.xml | 14.5 KB 14.5 KB | Display Display | EMDB header |
Images | emd_2832.tif | 758.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2832 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2832 | HTTPS FTP |
-Related structure data
Related structure data | 3j92MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar 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_2832.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of the rabbit 60S ribosomal subunit housing a defined nascent chain-P-site tRNA in complex with the E3 ligase Listerin and NEMF | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : rabbit 60S ribosomal subunit housing an in vitro translated nasce...
Entire | Name: rabbit 60S ribosomal subunit housing an in vitro translated nascent chain attached to a P-site tRNA in complex with the E3 ligase Listerin and NEMF. |
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Components |
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-Supramolecule #1000: rabbit 60S ribosomal subunit housing an in vitro translated nasce...
Supramolecule | Name: rabbit 60S ribosomal subunit housing an in vitro translated nascent chain attached to a P-site tRNA in complex with the E3 ligase Listerin and NEMF. type: sample / ID: 1000 / Number unique components: 5 |
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-Supramolecule #1: 60S ribosomal subunit
Supramolecule | Name: 60S ribosomal subunit / type: complex / ID: 1 / Recombinant expression: No Ribosome-details: ribosome-eukaryote: LSU 60S, LSU RNA 28S, LSU RNA 5.8S, LSU RNA 5S |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte / Location in cell: cytosol |
Molecular weight | Theoretical: 2.9 MDa |
-Macromolecule #1: transfer RNA
Macromolecule | Name: transfer RNA / type: rna / ID: 1 / Name.synonym: tRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: Sus scrofa (pig) / synonym: pig |
Molecular weight | Theoretical: 25 KDa |
-Macromolecule #2: nascent chain
Macromolecule | Name: nascent chain / type: protein_or_peptide / ID: 2 / Details: in vitro translated substrate / Number of copies: 1 / Recombinant expression: No |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 20 KDa |
-Macromolecule #3: Listerin
Macromolecule | Name: Listerin / type: protein_or_peptide / ID: 3 / Name.synonym: Ltn1, RNF160, ZNF294 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human / Location in cell: cytosol |
Molecular weight | Theoretical: 200 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1 |
Sequence | UniProtKB: E3 ubiquitin-protein ligase listerin |
-Macromolecule #4: NEMF
Macromolecule | Name: NEMF / type: protein_or_peptide / ID: 4 Name.synonym: nuclear export mediator factor, SDCCAG1, serologically defined colon cancer antigen 1 Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: human / Location in cell: cytosol |
Molecular weight | Theoretical: 120 KDa |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1 |
Sequence | UniProtKB: Ribosome quality control complex subunit NEMF |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.3 mg/mL |
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Buffer | pH: 7.4 / Details: 50 mM Hepes, 100 mM KAc, 5 mM MgAc2, 1 mM DTT |
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 for 3 seconds before plunging |
-Electron microscopy #1
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Microscopy ID | 1 |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification |
Date | Apr 30, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 30 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Electron microscopy #2
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Microscopy ID | 2 |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification |
Date | Aug 18, 2014 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 30 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 63826 |
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Details | RELION movement correction processing |
-Atomic model buiding 1
Initial model | PDB ID: 4w1z |
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Software | Name: Chimera, Coot |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT |
Output model | PDB-3j92: |