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- EMDB-2798: single-particle cryo reconstruction of the Large Ribosomal subuni... -

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Basic information

Entry
Database: EMDB / ID: EMD-2798
Titlesingle-particle cryo reconstruction of the Large Ribosomal subunit-associated protein Quality Control (RQC) complex in the context of Tae2 deletion
Map datareconstruction of RQC complex in the context of Tae2 protein deletion
Sample
  • Sample: Large 60S ribosomal subunit in complex with protein quality control components, in the context of Tae2 protein deletion
  • Complex: Large 60S ribosomal subunit
  • Protein or peptide: Large ribosomal subunit-associated quality control complexlex
KeywordsRING domain E3 ubiquitin ligase / translational surveillance / protein quality control / cryo-EM / listerin/Ltn1 / Tae2/Nemf
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.2 Å
AuthorsLyumkis D / Oliveira dos Passos D / Tahara EB / Webb K / Bennett EJ / Vinterbo S / Potter CS / Carragher B / Joazeiro CAP
CitationJournal: 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
DepositionOct 15, 2014-
Header (metadata) releaseOct 29, 2014-
Map releaseOct 29, 2014-
UpdateNov 26, 2014-
Current statusNov 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2798.png

Downloads & links

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Map

FileDownload / File: emd_2798.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of RQC complex in the context of Tae2 protein deletion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.38 Å/pix.
x 128 pix.
= 432.64 Å		3.38 Å/pix.
x 128 pix.
= 432.64 Å		3.38 Å/pix.
x 128 pix.
= 432.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.38 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-2.46616793 - 9.388854029999999
Average (Standard dev.)0.02034449 (±0.99826878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 432.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.383.383.38
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z432.640432.640432.640
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/NS128128128
D min/max/mean-2.4669.3890.020

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Supplemental data

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Sample components

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Entire : Large 60S ribosomal subunit in complex with protein quality contr...

EntireName: Large 60S ribosomal subunit in complex with protein quality control components, in the context of Tae2 protein deletion
Components
  • Sample: Large 60S ribosomal subunit in complex with protein quality control components, in the context of Tae2 protein deletion
  • Complex: Large 60S ribosomal subunit
  • Protein or peptide: Large ribosomal subunit-associated quality control complexlex

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Supramolecule #1000: Large 60S ribosomal subunit in complex with protein quality contr...

SupramoleculeName: Large 60S ribosomal subunit in complex with protein quality control components, in the context of Tae2 protein deletion
type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: Large 60S ribosomal subunit

SupramoleculeName: Large 60S ribosomal subunit / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: LSU 60S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741 / synonym: Baker's yeast
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: Large ribosomal subunit-associated quality control complexlex

MacromoleculeName: Large ribosomal subunit-associated quality control complexlex
type: protein_or_peptide / ID: 1 / Name.synonym: RQC / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741 / synonym: Baker's yeast

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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

Concentration0.1 mg/mL
BufferpH: 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
GridDetails: freshly plasma-cleaned holey carbon C-flat grid (Protochips) that had been overlaid with 2 nm thin carbon film
VitrificationCryogen 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.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected by monitoring power spectra of continuously acquired images in leginon
DateMay 22, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 2048 / Average electron dose: 32 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 92307 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailssee detailed methods in paper
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.2 Å / Resolution method: OTHER / Software - Name: Frealign / Number images used: 37819

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