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Entry
Database: EMDB / ID: EMD-6648
TitleEnsemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome
Map dataSaccharomyces cerevisiae 80S ribosome bound to eEF2-GDP-sordarin and TSV IRES, Structure I. For classification, 2D masking around the E site was used, including the 5' domain of PKI.
Sample
  • Sample: Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2
  • Complex: Ribosome
  • RNA: Internal Ribosome Entry Site
  • Protein or peptide: eukaryotic elongation factor 2EEF2
Keywordscryo-EM / translocation / IRES / ribosome / eEF2
Function / homology
Function and homology information


Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / Protein methylation / RMTs methylate histone arginines / positive regulation of translational fidelity / GDP-dissociation inhibitor activity ...Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / Protein methylation / RMTs methylate histone arginines / positive regulation of translational fidelity / GDP-dissociation inhibitor activity / mTORC1-mediated signalling / ribosome-associated ubiquitin-dependent protein catabolic process / Protein hydroxylation / : / pre-mRNA 5'-splice site binding / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Ribosomal scanning and start codon recognition / preribosome, small subunit precursor / translational elongation / response to cycloheximide / mRNA destabilization / Major pathway of rRNA processing in the nucleolus and cytosol / SRP-dependent cotranslational protein targeting to membrane / 90S preribosome / GTP hydrolysis and joining of the 60S ribosomal subunit / Formation of a pool of free 40S subunits / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of mRNA splicing, via spliceosome / protein-RNA complex assembly / ribosomal small subunit export from nucleus / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / translation regulator activity / ribosomal large subunit export from nucleus / G-protein alpha-subunit binding / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / positive regulation of protein kinase activity / rescue of stalled ribosome / translational termination / maturation of SSU-rRNA / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translation elongation factor activity / maturation of LSU-rRNA / ribosomal large subunit biogenesis / DNA-(apurinic or apyrimidinic site) endonuclease activity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Neutrophil degranulation / cellular response to amino acid starvation / ribosome assembly / small-subunit processome / protein kinase C binding / maintenance of translational fidelity / macroautophagy / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / modification-dependent protein catabolic process / ribosomal small subunit biogenesis / protein tag activity / small ribosomal subunit rRNA binding / rRNA processing / ribosomal small subunit assembly / cytoplasmic stress granule / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / ribosome biogenesis / 5S rRNA binding / cytoplasmic translation / small ribosomal subunit / cytosolic large ribosomal subunit / protein-folding chaperone binding / negative regulation of translation / protein ubiquitination / rRNA binding / ribosome / structural constituent of ribosome / positive regulation of protein phosphorylation / translation / G protein-coupled receptor signaling pathway / ribonucleoprotein complex / negative regulation of gene expression / response to antibiotic / mRNA binding / GTPase activity / ubiquitin protein ligase binding / GTP binding / nucleolus / mitochondrion / RNA binding / zinc ion binding / nucleoplasm / identical protein binding / metal ion binding / nucleus
Similarity search - Function
60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / : ...60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / : / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / Ribosomal protein S12e / 50S ribosomal protein L10, insertion domain superfamily / EF-G domain III/V-like / 60S ribosomal protein L10P, insertion domain / Small (40S) ribosomal subunit Asc1/RACK1 / Insertion domain in 60S ribosomal protein L10P / : / S27a-like superfamily / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal protein S25 / Ribosomal protein S26e signature. / Ribosomal protein S17e, conserved site / : / Tr-type G domain, conserved site / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S3, eukaryotic/archaeal / S25 ribosomal protein / Ribosomal protein L10e, conserved site / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein L10e / Ribosomal protein L27e, conserved site / Ribosomal protein S19A/S15e / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S30 / Ribosomal protein S3Ae, conserved site / Ribosomal protein S12e signature. / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal protein S27a / 40S ribosomal protein S11, N-terminal / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S6, eukaryotic / Ribosomal protein S7e / Ribosomal protein L1, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein S19e, conserved site / Ribosomal S17 / Ribosomal protein S19e signature. / Ribosomal protein L44e / Ribosomal protein L38e / Ribosomal protein S2, eukaryotic / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein L27e superfamily / Ribosomal protein S21e / Ribosomal protein S27, zinc-binding domain superfamily / Ribosomal L38e protein family / Ribosomal protein L29e / Ribosomal L29e protein family / 60S ribosomal protein L35 / Ribosomal protein L1 / Ribosomal protein S17, archaeal/eukaryotic / Ribosomal protein S27 / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S28e conserved site / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S28e / Ribosomal protein L35Ae, conserved site / 40S Ribosomal protein S10 / Ribosomal_S17 N-terminal / Ribosomal protein S3Ae / Ribosomal S3Ae family / Ribosomal protein L44 / Ribosomal protein S7e / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal L27e protein family / Ribosomal protein S5/S7, eukaryotic/archaeal / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein S6e / Plectin/S10, N-terminal / Ribosomal protein S13/S15, N-terminal / Ribosomal protein S15P / Plectin/S10 domain / Ribosomal S13/S15 N-terminal domain
Similarity search - Domain/homology
Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A ...Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS15 / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS11A / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Small ribosomal subunit protein uS8A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Small ribosomal subunit protein eS8A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL1A / Large ribosomal subunit protein uL2A / Small ribosomal subunit protein uS17A / Large ribosomal subunit protein eL18A / Small ribosomal subunit protein uS9A / Large ribosomal subunit protein uL11A / Small ribosomal subunit protein uS13A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Small ribosomal subunit protein eS32A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL18 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL13A / Small ribosomal subunit protein eS7A / Elongation factor 2 / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein eS27A / Large ribosomal subunit protein eL14A / Small ribosomal subunit protein RACK1 / Large ribosomal subunit protein eL32 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein eS26A / Small ribosomal subunit protein uS14A / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Small ribosomal subunit protein eS10A / Large ribosomal subunit protein eL13A / Small ribosomal subunit protein eS25A / Small ribosomal subunit protein eS28A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Taura syndrome virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsAbeyrathne PD / Koh CS / Grant T / Grigorieff N / Korostelev AA
CitationJournal: Elife / Year: 2016
Title: Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome.
Authors: Priyanka D Abeyrathne / Cha San Koh / Timothy Grant / Nikolaus Grigorieff / Andrei A Korostelev /
Abstract: Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the ...Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2•GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation.
History
DepositionMay 10, 2016-
Header (metadata) releaseJun 1, 2016-
Map releaseJun 1, 2016-
UpdateJun 1, 2016-
Current statusJun 1, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5juo
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6648.gif

Downloads & links

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Map

FileDownload / File: emd_6648.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSaccharomyces cerevisiae 80S ribosome bound to eEF2-GDP-sordarin and TSV IRES, Structure I. For classification, 2D masking around the E site was used, including the 5' domain of PKI.
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy EMDB: 0.032 / Movie #1: 0.027
Minimum - Maximum-0.06291012 - 0.12952782
Average (Standard dev.)-0.00331337 (±0.00947891)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z419.840419.840419.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0630.130-0.003

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 ...

EntireName: Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2
Components
  • Sample: Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2
  • Complex: Ribosome
  • RNA: Internal Ribosome Entry Site
  • Protein or peptide: eukaryotic elongation factor 2EEF2

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Supramolecule #1000: Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 ...

SupramoleculeName: Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2
type: sample / ID: 1000 / Number unique components: 3
Molecular weightTheoretical: 3.2 MDa

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightTheoretical: 3.2 MDa

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Macromolecule #1: Internal Ribosome Entry Site

MacromoleculeName: Internal Ribosome Entry Site / type: rna / ID: 1 / Name.synonym: IRES / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Taura syndrome virus
Molecular weightTheoretical: 80 KDa

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Macromolecule #2: eukaryotic elongation factor 2

MacromoleculeName: eukaryotic elongation factor 2 / type: protein_or_peptide / ID: 2 / Name.synonym: eEF2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightTheoretical: 94 KDa

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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

Concentration0.3 mg/mL
GridDetails: Quantifoil Cu 200 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 103 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 30487 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 18000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification.
Legacy - Electron beam tilt params: 0.7
Details10 electrons/physical pixel/second
DateNov 11, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 2662 / Average electron dose: 70 e/Å2
Details: Images recorded as movies in super-resolution mode.
Bits/pixel: 8
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: Unblur, CTFFIND4, FREALIGN
Details: The highest resolution included in the refinement was 7 Angstrom.
Number images used: 1105737
DetailsFREALIGN

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Atomic model buiding 1

Initial modelPDB ID:

3j6y

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5juo:
Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure I (fully rotated 40S subunit)

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