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- EMDB-2008: 3D reconstruction of a translating yeast 80S ribosome in complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2008
Title3D reconstruction of a translating yeast 80S ribosome in complex with Dom34p and Rli1p
Map dataThis is a 3D cryo-EM reconstruction of a translating yeast 80S ribosome in complex with Dom34p and Rli1p
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
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 9, 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
  • Download
  • Surface view colored by height
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMBD_2008.gif

Downloads & links

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Map

FileDownload / File: emd_2008.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D cryo-EM reconstruction of a translating yeast 80S ribosome in complex with Dom34p and Rli1p
Voxel sizeX=Y=Z: 1.17 Å
Density
Contour LevelBy AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum-0.94293511 - 1.85297704
Average (Standard dev.)0.0232901 (±0.15796183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 430.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.171.171.17
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z430.560430.560430.560
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-0.9431.8530.023

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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: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast

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Macromolecule #2: Dom34p

MacromoleculeName: Dom34p / type: protein_or_peptide / ID: 2 / Name.synonym: Pelota / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / 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

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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 75000
Sample stageSpecimen holder: autoloader / Specimen holder model: OTHER
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: 10000 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Wiener Filter
Final angle assignmentDetails: SPIDER
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
Number images used: 45700
Detailsmammalian Sec61 complex was added to saturate hydrophobic nascent polypeptide chains

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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

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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

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