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- EMDB-2010: 3D reconstruction of a translating yeast 80S ribosome in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2010
Title3D reconstruction of a translating yeast 80S ribosome in complex with Dom34p and Rli1p
Map dataThis map represents a stalled yeast 80S ribosome in complex with Dom34 and Rli1 before sorting for P-site tRNA.
Sample
  • Sample: Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem-loop mRNA in complex with Dom34p and Rli1p
  • Complex: S. cerevisiae 80S ribosome
  • Protein or peptide: Rli1p
  • Protein or peptide: Dom34p
Keywordstranslation / ribosome recycling / no-go mRNA decay
Function / homology
Function and homology information


RNA surveillance / Dom34-Hbs1 complex / nuclear-transcribed mRNA catabolic process, no-go decay / nuclear-transcribed mRNA catabolic process, non-stop decay / ribosome disassembly / mTORC1-mediated signalling / nonfunctional rRNA decay / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition ...RNA surveillance / Dom34-Hbs1 complex / nuclear-transcribed mRNA catabolic process, no-go decay / nuclear-transcribed mRNA catabolic process, non-stop decay / ribosome disassembly / mTORC1-mediated signalling / nonfunctional rRNA decay / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / Major pathway of rRNA processing in the nucleolus and cytosol / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal small subunit binding / 90S preribosome / positive regulation of translational initiation / ribosomal subunit export from nucleus / translational termination / RNA endonuclease activity / translation initiation factor activity / rescue of stalled ribosome / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / positive regulation of translation / meiotic cell cycle / small-subunit processome / translational initiation / cytoplasmic stress granule / rRNA processing / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / structural constituent of ribosome / iron ion binding / translation / cell division / nucleolus / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Translation release factor pelota / Pelota/DOM34, N-terminal domain / RLI, domain 1 / RLI1 / RNase L inhibitor RLI-like, possible metal-binding domain / Possible Fer4-like domain in RNase L inhibitor, RLI / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1/Pelota-like / eRF1 domain 3 ...Translation release factor pelota / Pelota/DOM34, N-terminal domain / RLI, domain 1 / RLI1 / RNase L inhibitor RLI-like, possible metal-binding domain / Possible Fer4-like domain in RNase L inhibitor, RLI / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 1 / eRF1 domain 3 / eRF1_1 / 4Fe-4S binding domain / 60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / Ribosomal protein S30 / Ribosomal protein S30 / : / Ribosomal protein S6, eukaryotic / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S6e signature. / Ribosomal protein S24e / Ribosomal protein S24e / Ribosomal protein S6e / Ribosomal protein S6e / Ribosomal protein S6e / Ribosomal protein L6, conserved site-2 / Ribosomal protein L6 signature 2. / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, RNA binding domain / Ribosomal protein L11/L12 / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / 50S ribosomal protein L30e-like / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L6 / Ribosomal protein L6 / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L14P, conserved site / Ribosomal protein L14 signature. / Ribosomal protein L14p/L23e / Ribosomal protein L14P / Ribosomal protein L14 superfamily / Ribosomal protein L14p/L23e / Ribosomal protein L23/L15e core domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL6A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS6A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL11A / Protein DOM34 / Translation initiation factor RLI1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsBecker T / Franckenberg S / Wickles S / Shoemaker CJ / Anger AM / Armache J-P / Sieber H / Ungewickell C / Berninghausen O / Daberkow I ...Becker T / Franckenberg S / Wickles S / Shoemaker CJ / Anger AM / Armache J-P / Sieber H / Ungewickell C / Berninghausen O / Daberkow I / Karcher A / Thomm M / Hopfner K-P / Green R / Beckmann R
CitationJournal: Nature / Year: 2012
Title: Structural basis of highly conserved ribosome recycling in eukaryotes and archaea.
Authors: Thomas Becker / Sibylle Franckenberg / Stephan Wickles / Christopher J Shoemaker / Andreas M Anger / Jean-Paul Armache / Heidemarie Sieber / Charlotte Ungewickell / Otto Berninghausen / Ingo ...Authors: Thomas Becker / Sibylle Franckenberg / Stephan Wickles / Christopher J Shoemaker / Andreas M Anger / Jean-Paul Armache / Heidemarie Sieber / Charlotte Ungewickell / Otto Berninghausen / Ingo Daberkow / Annette Karcher / Michael Thomm / Karl-Peter Hopfner / Rachel Green / Roland Beckmann /
Abstract: Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation ...Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.
History
DepositionDec 12, 2011-
Header (metadata) releaseFeb 15, 2012-
Map releaseFeb 17, 2012-
UpdateMar 1, 2012-
Current statusMar 1, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j16
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j16
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j16
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMBD_2010.gif

Downloads & links

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Map

FileDownload / File: emd_2010.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map represents a stalled yeast 80S ribosome in complex with Dom34 and Rli1 before sorting for P-site tRNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.24 Å/pix.
x 368 pix.
= 455.4 Å		1.24 Å/pix.
x 368 pix.
= 455.4 Å		1.24 Å/pix.
x 368 pix.
= 455.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum-0.78128618 - 1.73069727
Average (Standard dev.)0.0217575 (±0.14667007)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 455.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23751.23751.2375
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z455.400455.400455.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S213
start NC/NR/NS-184-184-183
NC/NR/NS368368368
D min/max/mean-0.7811.7310.022

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem...

EntireName: Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem-loop mRNA in complex with Dom34p and Rli1p
Components
  • Sample: Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem-loop mRNA in complex with Dom34p and Rli1p
  • Complex: S. cerevisiae 80S ribosome
  • Protein or peptide: Rli1p
  • Protein or peptide: Dom34p

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Supramolecule #1000: Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem...

SupramoleculeName: Saccharomyces cerevisiae 80S ribosome stalled by a synthetic stem-loop mRNA in complex with Dom34p and Rli1p
type: sample / ID: 1000
Oligomeric state: One 80S ribosome binds one Dom34p and one Rli1p
Number unique components: 3

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Supramolecule #1: S. cerevisiae 80S ribosome

SupramoleculeName: S. cerevisiae 80S ribosome / type: complex / ID: 1 / Name.synonym: Baker's yeast 80S ribosome / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast

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Macromolecule #1: Rli1p

MacromoleculeName: Rli1p / type: protein_or_peptide / ID: 1 / Name.synonym: ABCE1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pYES

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Macromolecule #2: Dom34p

MacromoleculeName: Dom34p / type: protein_or_peptide / ID: 2 / Name.synonym: Pelota / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pET28

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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
Details: 20 mM Tris/HCl pH 7.0, 150 mM KOAc, 10 mM Mg(OAc)2, 1.5 mM DTT, 0.005% Nikkol, 0.01 mg/ml Cycloheximide, 0.3% (w/v) Digitonin, 0.5 mM ADPNP
GridDetails: Quantifoil grids (3/3) with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blot for 10 seconds before plunging, use 2 layer of filter paper

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsFinal magnification of the object on the CCD image is 128200
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 5610 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 75000
Sample stageSpecimen holder: Dual tilt autoloader cartridge / Specimen holder model: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailsmammalian Sec61 complex was added to saturate hydrophobic nascent polypeptide chains
CTF correctionDetails: Wiener Filter
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: sorting for ribosome conformation and ligand presence was performed. This map represents the step after sorting for ligand presence but before sorting for p_site tRNA.
Number images used: 45700
Final angle assignmentDetails: SPIDER

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Atomic model buiding 1

Initial modelPDB ID:

3izq


Chain - Chain ID: 0
SoftwareName: MDFF
DetailsPDBEntryID_givenInChain. Protocol: rigid body followed by molecular dynamics flexible fitting. rigid body fitting of individual domains followed by molecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j16:
Models of ribosome-bound Dom34p and Rli1p and their ribosomal binding partners

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Atomic model buiding 2

Initial modelPDB ID:

3bk7


Chain - Chain ID: A
SoftwareName: MDFF
DetailsPDBEntryID_givenInChain. Protocol: rigid body followed by molecular dynamics flexible fitting. rigid body followed by molecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j16:
Models of ribosome-bound Dom34p and Rli1p and their ribosomal binding partners

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