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- EMDB-1809: Yeast 80S ribosome stalled by a stem-loop containing mRNA in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-1809
TitleYeast 80S ribosome stalled by a stem-loop containing mRNA in complex with Dom34p and N-terminally truncated Hbs1p.
Map dataThis is a 3D reconstruction of a yeast 80S ribosome stalled by stable stem-loop- containing mRNA in complex with proteins Dom34p and N-terminally-truncated Hbs1p (Hbs1-delta1-152).
Sample
  • Sample: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
  • 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: 27.0 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-
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: 0.22
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.22
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1809.gif

Downloads & links

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Map

FileDownload / File: emd_1809.map.gz / Format: CCP4 / Size: 9.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D reconstruction of a yeast 80S ribosome stalled by stable stem-loop- containing mRNA in complex with proteins Dom34p and N-terminally-truncated Hbs1p (Hbs1-delta1-152).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
3.62 Å/pix.
x 138 pix.
= 499.56 Å		3.62 Å/pix.
x 138 pix.
= 499.56 Å		3.62 Å/pix.
x 138 pix.
= 499.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.095 / Movie #1: 0.22
Minimum - Maximum-0.318632 - 1.41011
Average (Standard dev.)0.0360597 (±0.20508)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-69-69-68
Dimensions138138138
Spacing138138138
CellA=B=C: 499.56 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.623.623.62
M x/y/z138138138
origin x/y/z0.0000.0000.000
length x/y/z499.560499.560499.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-69-69-68
NX/NY/NZ138138138
MAP C/R/S213
start NC/NR/NS-69-69-68
NC/NR/NS138138138
D min/max/mean-0.3191.4100.036

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Supplemental data

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Sample components

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Entire : sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex

EntireName: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
Components
  • Sample: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
  • Complex: Saccharomyces cerevisiae 80S ribosome
  • Protein or peptide: Hbs1p
  • Protein or peptide: Dom34p

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Supramolecule #1000: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex

SupramoleculeName: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
type: sample / ID: 1000 / Details: Single particle / Oligomeric state: One ribosome / Number unique components: 3
Molecular weightExperimental: 3.3 MDa / Theoretical: 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
Details: Hbs1 is N-terminally truncated lacking the first 152 amino acids.
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers's yeast / Cell: Yeast cell / Organelle: Cytosol / Location in cell: Cytosol
Molecular weightExperimental: 69 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's yeast / Cell: Yeast cell / Organelle: Cytosol / 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, 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 layer of filter paper

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Electron microscopy

MicroscopeFEI TECNAI 12
TemperatureAverage: 84 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100000 times magnification
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Number real images: 97 / Average electron dose: 25 e/Å2 / Details: Images recorded on CCD. / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 90000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 90000
Sample stageSpecimen holder: FEI Polara Cartridge System / Specimen holder model: GATAN LIQUID NITROGEN

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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: 27.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 71400

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