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- EMDB-1342: Structures of modified eEF2 80S ribosome complexes reveal the rol... -

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Basic information

Entry
Database: EMDB / ID: EMD-1342
TitleStructures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation.
Map dataCryo-EM map of 80S ribosome bound with eEF2
Sample
  • Sample: Thermomyces lanuginosus 80S ribosome
  • Complex: Thermomyces lanuginosus
  • Protein or peptide: eEF2
  • RNA: tRNATransfer RNA
Function / homology
Function and homology information


Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / Protein methylation / translational elongation / translation elongation factor activity / Neutrophil degranulation / maintenance of translational fidelity / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome binding ...Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / Protein methylation / translational elongation / translation elongation factor activity / Neutrophil degranulation / maintenance of translational fidelity / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome binding / protein-folding chaperone binding / rRNA binding / ribonucleoprotein complex / GTPase activity / GTP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV ...Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / 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 / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor 2 / Elongation factor Tu-B
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Thermomyces lanuginosus (fungus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 11.3 Å
AuthorsTaylor DJ / Nilsson J / Merrill AR / Andersen GR / Nissen P / Frank J
CitationJournal: EMBO J / Year: 2007
Title: Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation.
Authors: Derek J Taylor / Jakob Nilsson / A Rod Merrill / Gregers Rom Andersen / Poul Nissen / Joachim Frank /
Abstract: On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 ...On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA-tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF-G in bacteria). We have obtained cryo-EM reconstructions of eukaryotic ribosomes complexed with ADP-ribosylated eEF2 (ADPR-eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase-coupled mechanism of translocation. Using the ADP-ribosyl group as a distinct marker, we observe conformational changes of ADPR-eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA-tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two-step model of translocation that begins with the eEF2/EF-G binding-induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA-tRNA complex from the decoding center so translocation of the mRNA-tRNA moiety may be completed by a head rotation of the small subunit.
History
DepositionMar 20, 2007-
Header (metadata) releaseMar 23, 2007-
Map releaseJun 12, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2p8w
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2p8w
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1342.map.gz / Format: CCP4 / Size: 32.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of 80S ribosome bound with eEF2
Voxel sizeX=Y=Z: 1.86 Å
Density
Contour Level1: 71.299999999999997 / Movie #1: 30
Minimum - Maximum-88.671499999999995 - 298.997000000000014
Average (Standard dev.)8.12074 (±27.037199999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-103-103-103
Dimensions205205205
Spacing205205205
CellA=B=C: 381.3 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.861.861.86
M x/y/z205205205
origin x/y/z0.0000.0000.000
length x/y/z381.300381.300381.300
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-103-103-103
NC/NR/NS205205205
D min/max/mean-88.672298.9978.121

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Supplemental data

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Sample components

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Entire : Thermomyces lanuginosus 80S ribosome

EntireName: Thermomyces lanuginosus 80S ribosome
Components
  • Sample: Thermomyces lanuginosus 80S ribosome
  • Complex: Thermomyces lanuginosus
  • Protein or peptide: eEF2
  • RNA: tRNATransfer RNA

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Supramolecule #1000: Thermomyces lanuginosus 80S ribosome

SupramoleculeName: Thermomyces lanuginosus 80S ribosome / type: sample / ID: 1000 / Number unique components: 3
Molecular weightTheoretical: 3.0 MDa

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Supramolecule #1: Thermomyces lanuginosus

SupramoleculeName: Thermomyces lanuginosus / type: complex / ID: 1 / Ribosome-details: ribosome-eukaryote: ALL
Molecular weightExperimental: 3.0 MDa

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Macromolecule #1: eEF2

MacromoleculeName: eEF2 / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 93 KDa

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Macromolecule #2: tRNA

MacromoleculeName: tRNA / type: rna / ID: 2 / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Thermomyces lanuginosus (fungus)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: see Methods
StainingType: NEGATIVE / Details: No staining (Cryo-EM)
GridDetails: Quanti-foil grids coated with a thin carbon layer
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 279 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Apply sample, wait 30s, blot for 6s, plunge in liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37642 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder: cartridge / Specimen holder model: OTHER
TemperatureAverage: 84 K
DateJul 7, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 93 / Average electron dose: 25 e/Å2 / Bits/pixel: 12
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correction of 3D map
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER package / Number images used: 93807

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: RSRef2.0
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 15 / Target criteria: cross-correlation
Output model

PDB-2p8w:
Fitted structure of eEF2 in the 80S:eEF2:GDPNP cryo-EM reconstruction

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