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- EMDB-1345: Structures of modified eEF2 80S ribosome complexes reveal the rol... -

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Basic information

Entry
Database: EMDB / ID: EMD-1345
TitleStructures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation.
Map dataCryo-EM map of the 80S ribosome:eEF2:GDPNP complex
Sample
  • Sample: Thermomyces lanuginosus 80S ribosome
  • Complex: Thermomyces lanuginosus
  • Protein or peptide: ADPR-eEF2
  • RNA: tRNA
Function / homology
Function and homology information


Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / Protein methylation / protein-synthesizing GTPase / translational elongation / translation elongation factor activity / Neutrophil degranulation / maintenance of translational fidelity / protein-folding chaperone binding ...Peptide chain elongation / Synthesis of diphthamide-EEF2 / positive regulation of translational elongation / Protein methylation / protein-synthesizing GTPase / translational elongation / translation elongation factor activity / Neutrophil degranulation / maintenance of translational fidelity / protein-folding chaperone binding / ribosome binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / rRNA binding / ribonucleoprotein complex / GTPase activity / GTP binding / identical protein binding / cytosol
Similarity search - Function
Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Elongation Factor G, domain II / Elongation Factor G, domain III / Translation elongation factor EFG/EF2, domain IV ...Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / 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 / 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: 8.9 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 23, 2007-
Header (metadata) releaseMar 23, 2007-
Map releaseJun 12, 2007-
UpdateAug 13, 2014-
Current statusAug 13, 2014Processing 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-2p8z, PDB-4v7h
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2p8z
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4v7h
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1345.gif

Downloads & links

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Map

FileDownload / File: emd_1345.map.gz / Format: CCP4 / Size: 32.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the 80S ribosome:eEF2:GDPNP complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.86 Å/pix.
x 205 pix.
= 381.3 Å		1.86 Å/pix.
x 205 pix.
= 381.3 Å		1.86 Å/pix.
x 205 pix.
= 381.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.86 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 70.400000000000006 / Movie #1: 30
Minimum - Maximum-103.710999999999999 - 280.857000000000028
Average (Standard dev.)7.70267 (±27.905999999999999)
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-103.711280.8577.703

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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: ADPR-eEF2
  • RNA: tRNA

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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 weightTheoretical: 3.0 MDa

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Macromolecule #1: ADPR-eEF2

MacromoleculeName: ADPR-eEF2 / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 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
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: 100 / Average electron dose: 25 e/Å2 / Bits/pixel: 12
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37642 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 39000
Sample stageSpecimen holder: cartridge / Specimen holder model: OTHER
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: 8.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER package / Number images used: 102689

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Atomic model buiding 1

Initial modelPDB ID:

1u2r


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-2p8z:
Fitted structure of ADPR-eEF2 in the 80S:ADPR-eEF2:GDPNP:sordarin cryo-EM reconstruction

PDB-4v7h:
Structure of the 80S rRNA and proteins and P/E tRNA for eukaryotic ribosome based on cryo-EM map of Thermomyces lanuginosus ribosome at 8.9A resolution

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