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- EMDB-5797: Structure of the Ribosome with Elongation Factor G Trapped in the... -

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Basic information

Entry
Database: EMDB / ID: EMD-5797
TitleStructure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State
Map dataRibosome reconstruction
Sample
  • Sample: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
  • Complex: 70S ribosome
  • Protein or peptide: Elongation Factor G
  • RNA: Transfer RNA
  • RNA: Messenger RNA
  • Ligand: Fusidic Acid
  • Ligand: Viomycin
Keywordsprotein structure / translation / EF-G / electron cryo-microscopy / single particle analysis
Function / homology
Function and homology information


intracellular anatomical structure / ribosome disassembly / guanosine tetraphosphate binding / translational elongation / translation elongation factor activity / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / GTPase activity / GTP binding / cytoplasm / cytosol
Similarity search - Function
Translation elongation factor EFG/EF2 / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / 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 ...Translation elongation factor EFG/EF2 / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / 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
Biological speciesEscherichia coli (E. coli) / synthetic construct (others) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsBrilot AF / Korostelev AA / Ermolenko DN / Grigorieff N
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Structure of the ribosome with elongation factor G trapped in the pretranslocation state.
Authors: Axel F Brilot / Andrei A Korostelev / Dmitri N Ermolenko / Nikolaus Grigorieff /
Abstract: During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by ...During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.
History
DepositionNov 19, 2013-
Header (metadata) releaseDec 18, 2013-
Map releaseDec 18, 2013-
UpdateJan 15, 2014-
Current statusJan 15, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5797_1.jpg

emd_5797_2.png

Downloads & links

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Map

FileDownload / File: emd_5797.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-0.71926343 - 1.93341196
Average (Standard dev.)-0.02908539 (±0.18453808)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.7191.933-0.029

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Supplemental data

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Sample components

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Entire : Post-translocation ribosome bound to EF-G with P, E sites occupie...

EntireName: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
Components
  • Sample: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
  • Complex: 70S ribosome
  • Protein or peptide: Elongation Factor G
  • RNA: Transfer RNA
  • RNA: Messenger RNA
  • Ligand: Fusidic Acid
  • Ligand: Viomycin

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Supramolecule #1000: Post-translocation ribosome bound to EF-G with P, E sites occupie...

SupramoleculeName: Post-translocation ribosome bound to EF-G with P, E sites occupied by tRNA
type: sample / ID: 1000 / Details: Sample was monodisperse / Number unique components: 6
Molecular weightTheoretical: 3 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 3 MDa

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Macromolecule #1: Elongation Factor G

MacromoleculeName: Elongation Factor G / type: protein_or_peptide / ID: 1 / Name.synonym: EF-G / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 78 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: K-12
SequenceUniProtKB: Elongation factor G
GO: translational elongation, GTP binding, translation elongation factor activity, intracellular anatomical structure
InterPro: Translation elongation factor EFG/EF2

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Macromolecule #2: Transfer RNA

MacromoleculeName: Transfer RNA / type: rna / ID: 2 / Name.synonym: tRNA / Details: Two tRNA present (P and E sites). / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightExperimental: 25 KDa

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Macromolecule #3: Messenger RNA

MacromoleculeName: Messenger RNA / type: rna / ID: 3 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12 KDa
SequenceString:
GGCAAGGAGG UAAAAAUGUU UAAACGUAAA UCUACU

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Macromolecule #4: Fusidic Acid

MacromoleculeName: Fusidic Acid / type: ligand / ID: 4 / Name.synonym: Fus / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 1 KDa
Chemical component information

ChemComp-FUA:
FUSIDIC ACID / antibiotic*YM

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Macromolecule #5: Viomycin

MacromoleculeName: Viomycin / type: ligand / ID: 5 / Name.synonym: Vio / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 1 KDa
Chemical component information


ChemComp, No image

ChemComp-PRD_000226:
Unknown entry

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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

Concentration0.4 mg/mL
BufferpH: 7.6
Details: 10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acid
GridDetails: C-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II
Method: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side ...Method: Freshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side port, blotted for 7 seconds with a positional offset of 2, and plunged into liquid ethane.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Automatically corrected using FEI software
DateNov 2, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14.0 µm / Number real images: 13341 / Average electron dose: 30 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 134615 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 6.95 µm / Nominal defocus min: 1.15 µm / Nominal magnification: 133333
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsRefinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details.
CTF correctionDetails: CTFFIND3, FREALIGN per micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: OTHER / Software - Name: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3
Details: Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details.
Number images used: 1341961

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