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- EMDB-1812: Yeast 80S ribosome stalled by a stem-loop containing mRNA in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-1812
TitleYeast 80S ribosome stalled by a stem-loop containing mRNA in complex with Dom34-Hbs1. The dataset is computationally sorted for absence of P-site tRNA and the presence of Dom34-Hbs1 complex.
Map dataThis is cryo-EM 3D reconstruction of a yeast 80S ribosome stalled by a stable stem-loop mRNA structure in complex with Dom34 and Hbs1. This 3D volume does NOT contain P-site tRNA.
Sample
  • Sample: Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 without P-site tRNA.
  • Complex: Saccharomyces cerevisiae 80S ribosome
  • Protein or peptide: Hbs1p
  • Protein or peptide: Dom34p
Keywordsribosome / stalling / mRNA / P-site tRNA / no-go mRNA decay
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.4 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 25, 2010-
Header (metadata) releaseMay 27, 2011-
Map releaseMay 27, 2011-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.48
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.48
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1812.gif

Downloads & links

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Map

FileDownload / File: emd_1812.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is cryo-EM 3D reconstruction of a yeast 80S ribosome stalled by a stable stem-loop mRNA structure in complex with Dom34 and Hbs1. This 3D volume does NOT contain P-site tRNA.
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy AUTHOR: 0.48 / Movie #1: 0.48
Minimum - Maximum-1.13045 - 2.14528
Average (Standard dev.)0.00536271 (±0.146684)
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-1.1302.1450.005

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Supplemental data

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Sample components

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Entire : Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 w...

EntireName: Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 without P-site tRNA.
Components
  • Sample: Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 without P-site tRNA.
  • Complex: Saccharomyces cerevisiae 80S ribosome
  • Protein or peptide: Hbs1p
  • Protein or peptide: Dom34p

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Supramolecule #1000: Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 w...

SupramoleculeName: Stem-loop stalled yeast 80S ribosome in complex with Dom34-Hbs1 without P-site tRNA.
type: sample / ID: 1000
Details: Mammalian Sec61 was added to saturate the hydrophobic signal sequence present in the nascent polypeptide chain.
Oligomeric state: one ribosome / Number unique components: 3
Molecular weightTheoretical: 3.3 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
Ribosome-details: ribosome-eukaryote: ALL
Molecular weightExperimental: 3.2 MDa / Theoretical: 3.2 MDa

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Macromolecule #1: Hbs1p

MacromoleculeName: Hbs1p / type: protein_or_peptide / ID: 1 / Name.synonym: Hbs1p / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers' yeast / Location in cell: cytosol
Molecular weightExperimental: 68 KDa / Theoretical: 68 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET28b

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Macromolecule #2: Dom34p

MacromoleculeName: Dom34p / type: protein_or_peptide / ID: 2 / Name.synonym: Dom34p / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers' yeast / Location in cell: cytosol
Molecular weightExperimental: 44 KDa / Theoretical: 44 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a

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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, 0.3 % Digitonin
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 layer 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: 3.78 µm / Nominal defocus min: 1.5 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: 78 / 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: 9.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: The dataset was sorted according to presence of Dom34-Hbs1 complex and absence of P-site tRNA.
Number images used: 38400
DetailsMammalian Sec61 complex was added to the sample to saturate the hydrophobic nascent chain

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Atomic model buiding 1

Initial modelPDB ID:

2vgm


Chain - Chain ID: A
SoftwareName: Molecular Dynamics based flexible fitting MDFF
DetailsPDBEntryID_givenInChain. Rigid body fitting of individual domains using Coot followed by MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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Atomic model buiding 2

Initial modelPDB ID:

2qi2

SoftwareName: Molecular Dynamics based flexible fitting MDFF
DetailsRigid body fitting of individual domains using Coot followed by MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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