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- EMDB-0055: Cryo-EM reconstruction of yeast 80S ribosome in complex with mRNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-0055
TitleCryo-EM reconstruction of yeast 80S ribosome in complex with mRNA, tRNA and eEF2 (GDP+AlF4/sordarin): SSU-focused map
Map data
Sample
  • Complex: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin)Eukaryotic ribosome
    • Complex: 80S ribosomeEukaryotic ribosome
    • Complex: mRNAMessenger RNA
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsPellegrino S / Yusupov M / Yusupova G / Hashem Y
CitationJournal: J Mol Biol / Year: 2018
Title: Structural Insights into the Role of Diphthamide on Elongation Factor 2 in mRNA Reading-Frame Maintenance.
Authors: Simone Pellegrino / Natalia Demeshkina / Eder Mancera-Martinez / Sergey Melnikov / Angelita Simonetti / Alexander Myasnikov / Marat Yusupov / Gulnara Yusupova / Yaser Hashem /
Abstract: One of the most critical steps of protein biosynthesis is the coupled movement of mRNA, which encodes genetic information, with tRNAs on the ribosome. In eukaryotes, this process is catalyzed by a ...One of the most critical steps of protein biosynthesis is the coupled movement of mRNA, which encodes genetic information, with tRNAs on the ribosome. In eukaryotes, this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-electron microscopy structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights into the role of diphthamide in the mechanism of translation fidelity in eukaryotes.
History
DepositionJun 14, 2018-
Header (metadata) releaseJul 11, 2018-
Map releaseJul 11, 2018-
UpdateApr 1, 2020-
Current statusApr 1, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0055.png

Downloads & links

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Map

FileDownload / File: emd_0055.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.16047029 - 0.308272
Average (Standard dev.)0.0010391705 (±0.010803084)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z396.000396.000396.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.1600.3080.001

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Supplemental data

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Sample components

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Entire : 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of G...

EntireName: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin)Eukaryotic ribosome
Components
  • Complex: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin)Eukaryotic ribosome
    • Complex: 80S ribosomeEukaryotic ribosome
    • Complex: mRNAMessenger RNA

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Supramolecule #1: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of G...

SupramoleculeName: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Molecular weightTheoretical: 3.4 MDa

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Supramolecule #2: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#81, #83-#84
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: mRNA

SupramoleculeName: mRNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #82
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: synthetic construct (others)

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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

BufferpH: 7.5
Component:
ConcentrationFormula
10.0 mMHepes-KOH
5.0 mMMg(OAc)2
40.0 mMKOAc
10.0 mMNH4Cl
1.0 mMDTT
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 4, blot waiting time 30 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4ug0

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 77200

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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