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- EMDB-2702: Cryo-EM structure of the mammalian 60S ribosomal subunit in compl... -

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Basic information

Entry
Database: EMDB / ID: EMD-2702
TitleCryo-EM structure of the mammalian 60S ribosomal subunit in complex with eIF6 and Listerin
Map data60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
Sample
  • Sample: mammalian 60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
  • Complex: 60S ribosomal subunit
  • Protein or peptide: eukaryotic initiation factor 6
  • Protein or peptide: Listerin
Keywordsribosome / ubiquitination / quality control
Biological speciesOryctolagus cuniculus (rabbit) / Saccharomyces cerevisiae (brewer's yeast) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsShao S / Hegde RS
CitationJournal: 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
DepositionJul 8, 2014-
Header (metadata) releaseJul 23, 2014-
Map releaseAug 27, 2014-
UpdateOct 1, 2014-
Current statusOct 1, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2702.tif

Downloads & links

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Map

FileDownload / File: emd_2702.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å		1.34 Å/pix.
x 320 pix.
= 428.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.11377499 - 0.25224671
Average (Standard dev.)0.00223281 (±0.01502677)
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-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1140.2520.002

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Supplemental data

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Sample components

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Entire : mammalian 60S ribosomal subunit in complex with eIF6 and Listerin...

EntireName: mammalian 60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
Components
  • Sample: mammalian 60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
  • Complex: 60S ribosomal subunit
  • Protein or peptide: eukaryotic initiation factor 6
  • Protein or peptide: Listerin

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Supramolecule #1000: mammalian 60S ribosomal subunit in complex with eIF6 and Listerin...

SupramoleculeName: mammalian 60S ribosomal subunit in complex with eIF6 and Listerin E3 ligase
type: sample / ID: 1000 / Number unique components: 3

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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: cytoplasm
Molecular weightTheoretical: 2.9 MDa

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Macromolecule #1: eukaryotic initiation factor 6

MacromoleculeName: eukaryotic initiation factor 6 / type: protein_or_peptide / ID: 1 / Name.synonym: eIF6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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Macromolecule #2: Listerin

MacromoleculeName: Listerin / type: protein_or_peptide / ID: 2 / Name.synonym: RNF160, ZNF294 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 200 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1

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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

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 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateApr 1, 2014
Image recordingCategory: 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsRELION movement correction processing
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 9148

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