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- EMDB-5799: Structure of the Ribosome with Elongation Factor G Trapped in the... -

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Entry
Database: EMDB / ID: EMD-5799
TitleStructure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State
Map dataReconstruction of a pre-translocation ribosome
Sample
  • Sample: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
  • Complex: 70S ribosome
  • RNA: Transfer RNA
  • RNA: Messenger RNA
  • Ligand: Viomycin
Keywordsprotein structure / translation / EF-G / electron cryo-microscopy / single particle analysis
Function / homology
Function and homology information


stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis ...stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / large ribosomal subunit rRNA binding / transferase activity / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site ...Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / : / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L16 signature 1. / Ribosomal protein L10P / Ribosomal protein L10 / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, RNA binding domain / Ribosomal protein L11/L12 / Ribosomal protein L36 signature. / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / : / : / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L20 signature. / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L14P, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein S2, bacteria/mitochondria/plastid
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 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-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4v7c
  • Surface level: 0.32
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5799_1.jpg

emd_5799_2.png

Downloads & links

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Map

FileDownload / File: emd_5799.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a pre-translocation ribosome
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: 0.32 / Movie #1: 0.32
Minimum - Maximum-0.46019435 - 1.30163205
Average (Standard dev.)-0.02749274 (±0.17507136)
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-0.4601.302-0.027

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Supplemental data

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Sample components

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Entire : Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA

EntireName: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
Components
  • Sample: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
  • Complex: 70S ribosome
  • RNA: Transfer RNA
  • RNA: Messenger RNA
  • Ligand: Viomycin

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Supramolecule #1000: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA

SupramoleculeName: Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
type: sample / ID: 1000 / Details: Sample was monodisperse. / Number unique components: 4
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: Transfer RNA

MacromoleculeName: Transfer RNA / type: rna / ID: 1 / Name.synonym: tRNA / 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 #2: Messenger RNA

MacromoleculeName: Messenger RNA / type: rna / ID: 2 / 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 #3: Viomycin

MacromoleculeName: Viomycin / type: ligand / ID: 3 / 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: 7.6 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: 85115

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Atomic model buiding 1

Initial modelPDB ID:

4gd1
PDB Unreleased entry

SoftwareName: Chimera, CNS
DetailsChain Y not used in refinement. See primary citation Supplementary Information for details.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-4v7c:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA structure)

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Atomic model buiding 2

Initial modelPDB ID:

3r8s
PDB Unreleased entry

SoftwareName: Chimera, CNS
DetailsSee primary citation Supplementary Information for details.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-4v7c:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA structure)

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Atomic model buiding 3

Initial modelPDB ID:

1mms

SoftwareName: Chimera, CNS
DetailsUsed to rebuild the L11 stalk.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-4v7c:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA structure)

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Atomic model buiding 4

Initial modelPDB ID:

3u4m

SoftwareName: Chimera, CNS
DetailsUsed to build homology model for L1.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-4v7c:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA structure)

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Atomic model buiding 5

Initial modelPDB ID:

1zav

SoftwareName: Chimera, CNS
DetailsUsed to build homology model for L10.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-4v7c:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA structure)

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