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- EMDB-1808: Yeast 80S ribosome stalled by a stem-loop containing mRNA. -

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Basic information

Entry
Database: EMDB / ID: EMD-1808
TitleYeast 80S ribosome stalled by a stem-loop containing mRNA.
Map dataThis is a map of yeast 80S ribosome stalled by a stable 66 nucleotide stem-loop mRNA structure (Hosoda et al., JBC 2003). It contains a P-site tRNA.
Sample
  • Sample: Stem-loop stalled yeast 80S ribosome
  • Complex: Saccharomyces cerevisiae 80S ribosome
KeywordsRibosome / stalling / mRNA / P-site tRNA / no-go mRNA decay
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.1 Å
AuthorsBecker T / Armache JP / Anger AM / Jarasch A / Villa E / Sieber H / AbdelMotaal B / Berninghausen O / Mielke T / Beckmann R
CitationJournal: Nat Struct Mol Biol / Year: 2011
Title: Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.
Authors: Thomas Becker / Jean-Paul Armache / Alexander Jarasch / Andreas M Anger / Elizabeth Villa / Heidemarie Sieber / Basma Abdel Motaal / Thorsten Mielke / Otto Berninghausen / Roland Beckmann /
Abstract: No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. ...No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34-Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1-eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.
History
DepositionOct 23, 2010-
Header (metadata) releaseMay 27, 2011-
Map releaseMay 27, 2011-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.29
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.29
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1808.gif

Downloads & links

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Map

FileDownload / File: emd_1808.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of yeast 80S ribosome stalled by a stable 66 nucleotide stem-loop mRNA structure (Hosoda et al., JBC 2003). It contains a P-site tRNA.
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy AUTHOR: 0.29 / Movie #1: 0.29
Minimum - Maximum-0.978761 - 1.87877
Average (Standard dev.)0.0109003 (±0.140744)
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 °

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-0.9791.8790.011

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Supplemental data

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Sample components

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Entire : Stem-loop stalled yeast 80S ribosome

EntireName: Stem-loop stalled yeast 80S ribosome
Components
  • Sample: Stem-loop stalled yeast 80S ribosome
  • Complex: Saccharomyces cerevisiae 80S ribosome

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Supramolecule #1000: Stem-loop stalled yeast 80S ribosome

SupramoleculeName: Stem-loop stalled yeast 80S ribosome / type: sample / ID: 1000 / Details: Single particle / Oligomeric state: One ribosome / Number unique components: 1
Molecular weightTheoretical: 3.2 MDa

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Supramolecule #1: Saccharomyces cerevisiae 80S ribosome

SupramoleculeName: Saccharomyces cerevisiae 80S ribosome / type: complex / ID: 1 / Name.synonym: Yeast 80S ribosome
Details: The mRNA stem-loop structure is not visible in the Cryo-EM reconstruction indicating its flexibility
Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 3.2 MDa / Theoretical: 3.2 MDa

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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

Concentration0.02 mg/mL
BufferpH: 7
Details: 20 mM Tris/HCl, pH 7.0, 80 mM NaCl, 97 mM KOAc, 10 mM Mg(OAc)2, 1.5 mM DTT, 0.02 % Nikkol, 1.8 % Glycerol, 0.01 mg/ml Cycloheximide, 500 0.5 mM GDPNP
GridDetails: Quantifoil Grid with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blotted for 10 seconds before plunging, used 2 layers of filter paper

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 39000
Sample stageSpecimen holder: FEI Polara Cartridge System / Specimen holder model: OTHER
TemperatureAverage: 84 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100000 times magnification
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 4.76 µm / Number real images: 41 / Average electron dose: 25 e/Å2
Details: Scanned with a Heidelberg PrimeScan drum scanner at 5334 dpi
Od range: 1.2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correction on the level of 3D volumes (SPIDER TF CTS command)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 31400

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