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- EMDB-2832: Cryo-EM map of a mammalian 60S ribosome-nascent chain-tRNA comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-2832
TitleCryo-EM map of a mammalian 60S ribosome-nascent chain-tRNA complex with Listerin and NEMF
Map datareconstruction of the rabbit 60S ribosomal subunit housing a defined nascent chain-P-site tRNA in complex with the E3 ligase Listerin and NEMF
Sample
  • Sample: 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.
  • Complex: 60S ribosomal subunit
  • RNA: transfer RNA
  • Protein or peptide: nascent chain
  • Protein or peptide: Listerin
  • Protein or peptide: NEMFNew England Motor Freight
Keywordsribosome-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
NFACT protein, C-terminal / NFACT protein C-terminal domain / E3 ubiquitin-protein ligase listerin / Listerin, zinc finger, RING-type / Zinc finger, RING-CH-type / The RING-variant domain is a C4HC3 zinc-finger like motif found in a number of cellular and viral proteins. Some of these proteins have been shown both in vivo and in vitro to have ubiquitin E3 ligase activity. / NFACT, RNA-binding domain / NFACT protein RNA binding domain / NFACT N-terminal and middle domains / Zinc finger RING-type profile. ...NFACT protein, C-terminal / NFACT protein C-terminal domain / E3 ubiquitin-protein ligase listerin / Listerin, zinc finger, RING-type / Zinc finger, RING-CH-type / The RING-variant domain is a C4HC3 zinc-finger like motif found in a number of cellular and viral proteins. Some of these proteins have been shown both in vivo and in vitro to have ubiquitin E3 ligase activity. / NFACT, RNA-binding domain / NFACT protein RNA binding domain / NFACT N-terminal and middle domains / Zinc finger RING-type profile. / Zinc finger, RING-type / Armadillo-like helical / Armadillo-type fold / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Ribosome quality control complex subunit NEMF / E3 ubiquitin-protein ligase listerin
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Sus scrofa (pig) / unidentified (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsShao S / Brown A / Santhanam B / Hegde RS
CitationJournal: 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
DepositionDec 2, 2014-
Header (metadata) releaseJan 21, 2015-
Map releaseJan 21, 2015-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j92
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2832.tif

Downloads & links

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Map

FileDownload / File: emd_2832.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of the rabbit 60S ribosomal subunit housing a defined nascent chain-P-site tRNA in complex with the E3 ligase Listerin and NEMF
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.23654813 - 0.49433696
Average (Standard dev.)0.0025526 (±0.01947696)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2370.4940.003

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Supplemental data

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Sample components

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Entire : rabbit 60S ribosomal subunit housing an in vitro translated nasce...

EntireName: 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.
Components
  • Sample: 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.
  • Complex: 60S ribosomal subunit
  • RNA: transfer RNA
  • Protein or peptide: nascent chain
  • Protein or peptide: Listerin
  • Protein or peptide: NEMFNew England Motor Freight

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Supramolecule #1000: rabbit 60S ribosomal subunit housing an in vitro translated nasce...

SupramoleculeName: 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

SupramoleculeName: 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
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte / Location in cell: cytosol
Molecular weightTheoretical: 2.9 MDa

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Macromolecule #1: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 1 / Name.synonym: tRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Sus scrofa (pig) / synonym: pig
Molecular weightTheoretical: 25 KDa

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Macromolecule #2: nascent chain

MacromoleculeName: nascent chain / type: protein_or_peptide / ID: 2 / Details: in vitro translated substrate / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 20 KDa

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Macromolecule #3: Listerin

MacromoleculeName: Listerin / type: protein_or_peptide / ID: 3 / Name.synonym: Ltn1, RNF160, ZNF294 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: cytosol
Molecular weightTheoretical: 200 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1
SequenceUniProtKB: E3 ubiquitin-protein ligase listerin

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Macromolecule #4: NEMF

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human) / synonym: human / Location in cell: cytosol
Molecular weightTheoretical: 120 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1
SequenceUniProtKB: Ribosome quality control complex subunit NEMF

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.4 / Details: 50 mM Hepes, 100 mM KAc, 5 mM MgAc2, 1 mM DTT
GridDetails: Quantifoil R2/2 on 400 mesh Cu grid with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: Blot for 3 seconds before plunging

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Electron microscopy #1

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID1
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateApr 30, 2014
Image recordingCategory: 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

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Electron microscopy #2

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Microscopy ID2
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateAug 18, 2014
Image recordingCategory: 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

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 63826
DetailsRELION movement correction processing

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Atomic model buiding 1

Initial modelPDB ID:

4w1z
PDB Unreleased entry

SoftwareName: Chimera, Coot
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

PDB-3j92:
Structure and assembly pathway of the ribosome quality control complex

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Atomic model buiding 2

Initial modelPDB ID:

4w20
PDB Unreleased entry

SoftwareName: Chimera, Coot
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

PDB-3j92:
Structure and assembly pathway of the ribosome quality control complex

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