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Yorodumi- EMDB-1809: Yeast 80S ribosome stalled by a stem-loop containing mRNA in comp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1809 | |||||||||
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Title | Yeast 80S ribosome stalled by a stem-loop containing mRNA in complex with Dom34p and N-terminally truncated Hbs1p. | |||||||||
Map data | This 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 |
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Keywords | Ribosome / stalling / mRNA / P-site tRNA / no-go mRNA decay | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 27.0 Å | |||||||||
Authors | Becker T / Armache JP / Anger AM / Jarasch A / Villa E / Sieber H / AbdelMotaal B / Berninghausen O / Mielke T / Beckmann R | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1809.map.gz | 1.2 MB | EMDB map data format | |
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Header (meta data) | emd-1809-v30.xml emd-1809.xml | 12 KB 12 KB | Display Display | EMDB header |
Images | EMD-1809.gif | 166.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1809 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1809 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_1809.map.gz / Format: CCP4 / Size: 9.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This 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). | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.62 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
Entire | Name: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex |
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Components |
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-Supramolecule #1000: sSem-loop stalled yeast 80S ribosome/Dom34-delta(1-152)Hbs1 complex
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 3.3 MDa / Theoretical: 3.3 MDa |
-Supramolecule #1: Saccharomyces cerevisiae 80S ribosome
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 3.2 MDa / Theoretical: 3.2 MDa |
-Macromolecule #1: Hbs1p
Macromolecule | Name: 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 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers's yeast / Cell: Yeast cell / Organelle: Cytosol / Location in cell: Cytosol |
Molecular weight | Experimental: 69 KDa / Theoretical: 68 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET28b |
-Macromolecule #2: Dom34p
Macromolecule | Name: Dom34p / type: protein_or_peptide / ID: 2 / Name.synonym: Dom34p / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers's yeast / Cell: Yeast cell / Organelle: Cytosol / Location in cell: Cytosol |
Molecular weight | Experimental: 44 KDa / Theoretical: 44 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.02 mg/mL |
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Buffer | pH: 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 |
Grid | Details: Quantifoil Grid with 2 nm carbon on top |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot Method: Blotted for 10 seconds before plunging, used 2 layer of filter paper |
-Electron microscopy
Microscope | FEI TECNAI 12 |
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Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 90000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 90000 |
Sample stage | Specimen holder: FEI Polara Cartridge System / Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Average: 84 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100000 times magnification |
Image recording | Category: 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 |
-Image processing
CTF correction | Details: CTF correction on the level of 3D volumes (SPIDER TF CTS command) |
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Final reconstruction | Applied 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 |