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- EMDB-6173: Electron cryo-microscopy of peptidyl-(~A/P)tRNA-60S particles, ES27out -

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Basic information

Entry
Database: EMDB / ID: EMD-6173
TitleElectron cryo-microscopy of peptidyl-(~A/P)tRNA-60S particles, ES27out
Map datapeptidyl-tRNA-60S; tRNA in an A-A/P hybrid position
Sample
  • Sample: RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG peptide
  • Complex: 60S ribosome
Keywordsribosome quality control complex / RQC / eukaryotic ribosome rescue / stalled nascent chain
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsShen PS / Park J / Qin Y / Li X / Parsawar K / Larson M / Cox J / Cheng Y / Lambowitz AM / Weissman JS ...Shen PS / Park J / Qin Y / Li X / Parsawar K / Larson M / Cox J / Cheng Y / Lambowitz AM / Weissman JS / Brandman O / Frost A
CitationJournal: Science / Year: 2015
Title: Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.
Authors: Peter S Shen / Joseph Park / Yidan Qin / Xueming Li / Krishna Parsawar / Matthew H Larson / James Cox / Yifan Cheng / Alan M Lambowitz / Jonathan S Weissman / Onn Brandman / Adam Frost /
Abstract: In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report ...In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").
History
DepositionNov 4, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 9, 2015-
UpdateJan 13, 2016-
Current statusJan 13, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6173.gif

Downloads & links

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Map

FileDownload / File: emd_6173.map.gz / Format: CCP4 / Size: 34.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpeptidyl-tRNA-60S; tRNA in an A-A/P hybrid position
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.44 Å/pix.
x 210 pix.
= 512.4 Å		2.44 Å/pix.
x 210 pix.
= 512.4 Å		2.44 Å/pix.
x 210 pix.
= 512.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.44 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.03816437 - 0.11244388
Average (Standard dev.)0.00031911 (±0.00686892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 512.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.442.442.44
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z512.400512.400512.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-189
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-0.0380.1120.000

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Supplemental data

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Sample components

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Entire : RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG ...

EntireName: RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG peptide
Components
  • Sample: RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG peptide
  • Complex: 60S ribosome

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Supramolecule #1000: RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG ...

SupramoleculeName: RQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG peptide
type: sample / ID: 1000
Details: RQC particles were 3D classified to reveal distinct subclasses containing various RQC components.
Number unique components: 1

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Supramolecule #1: 60S ribosome

SupramoleculeName: 60S ribosome / type: complex / ID: 1 / Name.synonym: large ribosomal subunit / Recombinant expression: No / Database: NCBI
Ribosome-details: ribosome-eukaryote: LSU 60S, LSU RNA 28S, LSU RNA 5.8S, LSU RNA 5S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741 / synonym: 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

BufferpH: 7.4 / Details: 100 mM KOAc, 10 mM MgCl2, 25 mM HEPES-KOH
GridDetails: 200 mesh Quantifoil R2/2 grid
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Specialist opticsEnergy filter - Name: Gatan
DetailsUCSF Image4 on-the-fly motion correction
DateJul 22, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2000 / Average electron dose: 35 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 31000
Sample stageSpecimen holder: LN2 cooled / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected using the semi-automated swarm tool in e2boxer.py of the EMAN2 package. All 2D and 3D processing was performed in RELION.
CTF correctionDetails: each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: RELION, CTFFIND3
Details: Micrographs were motion-corrected via the UCSFImage4 package.
Number images used: 15800

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