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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: 0055
TitleCryo-EM reconstruction of yeast 80S ribosome in complex with mRNA, tRNA and eEF2 (GDP+AlF4/sordarin): SSU-focused map
Map data
Sample80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin):
80S ribosome / mRNAMessenger RNA
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 4.3 Å resolution
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.
DateDeposition: Jun 14, 2018 / Header (metadata) release: Jul 11, 2018 / Map release: Jul 11, 2018 / Last update: Aug 8, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6gqb
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_0055.map.gz (map file in CCP4 format, 186625 KB)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
360 pix
1.1 Å/pix.
= 396. Å
360 pix
1.1 Å/pix.
= 396. Å
360 pix
1.1 Å/pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour Level:0.035 (by author), 0.035 (movie #1):
Minimum - Maximum-0.16047029 - 0.308272
Average (Standard dev.)0.0010391705 (0.010803084)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions360360360
Origin0.0.0.
Limit359.359.359.
Spacing360360360
CellA=B=C: 396.0 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
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)
Number of components: 3

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Component #1: protein, 80S ribosome in complex with mRNA, tRNA and eEF2 in pres...

ProteinName: 80S ribosome in complex with mRNA, tRNA and eEF2 in presence of GDP, AlF4 and anti-fungal drug (sordarin)
Recombinant expression: No
MassTheoretical: 3.4 MDa

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Component #2: protein, 80S ribosome

ProteinName: 80S ribosome / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, mRNA

ProteinName: mRNAMessenger RNA / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 % / Details: blot force 4, blot waiting time 30 s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 6 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 77200
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL

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