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- EMDB-6171: Electron cryo-microscopy of peptidyl-tRNA bound to yeast 60S ribosome -

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Basic information

Entry
Database: EMDB / ID: 6171
TitleElectron cryo-microscopy of peptidyl-tRNA bound to yeast 60S ribosome
Map datastructure of peptidyl-tRNA-60S, related to Fig. 1A of the primary citation. For the masked, sharpened map, see EMD-6201
SampleRQC particles purified by co-IP of Rqc1-FLAG, eluted with 3xFLAG peptide:
ribosome-eukaryote
Keywordsribosome quality control complex / RQC / eukaryotic ribosome rescue / stalled nascent chain
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 5.9 Å resolution
AuthorsShen 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").
DateDeposition: Nov 4, 2014 / Header (metadata) release: Dec 24, 2014 / Map release: Jan 7, 2015 / Last update: Jan 21, 2015

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0058
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0058
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6171.map.gz (map file in CCP4 format, 289408 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
420 pix
1.22 Å/pix.
= 512.4 Å
420 pix
1.22 Å/pix.
= 512.4 Å
420 pix
1.22 Å/pix.
= 512.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.22 Å
Density
Contour Level:0.0058 (by author), 0.0058 (movie #1):
Minimum - Maximum-0.01445245 - 0.04920224
Average (Standard dev.)9.576E-5 (0.00220581)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions420420420
Origin000
Limit419419419
Spacing420420420
CellA=B=C: 512.4 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.221.221.22
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z512.400512.400512.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0140.0490.000

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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
Details: RQC particles were 3D classified to reveal distinct subclasses containing various RQC components.
Number of components: 1

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Component #1: ribosome-eukaryote, 60S ribosome

Ribosome-eukaryoteName: 60S ribosome / a.k.a: large ribosomal subunit / Eukaryote: LSU 60S, LSU RNA 28S, LSU RNA 5.8S, LSU RNA 5S / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: BY4741

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 100 mM KOAc, 10 mM MgCl2, 25 mM HEPES-KOH / pH: 7.4
Support film200 mesh Quantifoil R2/2 grid + lacey carbon grid with ultrathin carbon
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 90 K / Humidity: 75 % / Method: Blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Jul 22, 2013 / Details: UCSF Image4 on-the-fly motion correction
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 35 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 800 - 2000 nm / Energy filter: Gatan
Specimen HolderHolder: LN2 cooled / Model: GATAN LIQUID NITROGEN / Temperature: 85 K ( 80 - 90 K)
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3459 / Bit depth: 8

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 29956
Details: Particles were selected using the semi-automated swarm tool in e2boxer.py of the EMAN2 package. All 2D and 3D processing was performed in RELION.
3D reconstructionSoftware: RELION, CTFFIND3 / CTF correction: each particle
Details: Unmasked map related to Fig. 1A of the primary citation. For the masked, sharpened map, see EMD-6201
Resolution: 5.9 Å / Resolution method: FSC 0.143, gold-standard

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