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- EMDB-5775: Cryo-EM reconstruction of a authentic 70S-tRNA2-mRNA-EF-G translo... -

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Basic information

Entry
Database: EMDB / ID: EMD-5775
TitleCryo-EM reconstruction of a authentic 70S-tRNA2-mRNA-EF-G translocation intermediate
Map datareconstruction of an in vitro prepared 70S-tRNA2-mRNA-EF-G complex
Sample
  • Sample: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Elongation Factor GEF-G
  • RNA: modified formyl-methionine specific initiator transfer RNA
  • RNA: formyl-methionine specific initiator transfer RNA
  • RNA: messenger RNA
Keywordsribosome / translocation / tRNAs / mRNA / EF-G / head-swivel
Function / homology
Function and homology information


ribosome disassembly / guanosine tetraphosphate binding / translational elongation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding ...ribosome disassembly / guanosine tetraphosphate binding / translational elongation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, 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 / translational termination / DnaA-L2 complex / negative regulation of translational initiation / four-way junction DNA binding / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / translation elongation factor activity / regulation of mRNA stability / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / transcription elongation factor complex / positive regulation of RNA splicing / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / DNA endonuclease activity / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / : / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / regulation of translation / ribosome biogenesis / ribosome binding / large ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic large ribosomal subunit / small ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / molecular adaptor activity / ribosome / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / mRNA binding / negative regulation of DNA-templated transcription / GTP binding / protein homodimerization activity / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
: / Translation elongation factor EFG/EF2 / Elongation factor G, domain III / EFG, domain V / Ribosomal protein L7/L12, oligomerisation / Ribosomal protein L7/L12, oligomerisation domain superfamily / Ribosomal protein L7/L12 dimerisation domain / Ribosomal protein L7/L12 / Ribosomal protein L7/L12, C-terminal / Ribosomal protein L7/L12 C-terminal domain ...: / Translation elongation factor EFG/EF2 / Elongation factor G, domain III / EFG, domain V / Ribosomal protein L7/L12, oligomerisation / Ribosomal protein L7/L12, oligomerisation domain superfamily / Ribosomal protein L7/L12 dimerisation domain / Ribosomal protein L7/L12 / Ribosomal protein L7/L12, C-terminal / Ribosomal protein L7/L12 C-terminal domain / Ribosomal protein L7/L12, C-terminal/adaptor protein ClpS-like / 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 / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / 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 / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Elongation factor Tu domain 2 / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L11, bacterial-type / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein S21 / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / Ribosomal protein L10-like domain superfamily / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L10 / Ribosomal protein L10P / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L17 signature. / 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 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 L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / 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 L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein S6, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6 signature.
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Elongation factor G / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL12 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Elongation factor G / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL12 / Large ribosomal subunit protein bL19 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsRamrath DJF / Lancaster L / Sprink T / Mielke T / Loerke J / Noller HF / Spahn CMT
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation.
Authors: David J F Ramrath / Laura Lancaster / Thiemo Sprink / Thorsten Mielke / Justus Loerke / Harry F Noller / Christian M T Spahn /
Abstract: During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is ...During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.
History
DepositionOct 24, 2013-
Header (metadata) releaseNov 20, 2013-
Map releaseDec 11, 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: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v7b
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5775.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of an in vitro prepared 70S-tRNA2-mRNA-EF-G complex
Voxel sizeX=Y=Z: 1.26 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-5.07783175 - 12.70241165
Average (Standard dev.)0.1971412 (±0.87503892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.261.261.26
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-5.07812.7020.197

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Supplemental data

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Sample components

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Entire : ribosomal 70S-EF-G complex at an authentic intermediate state dur...

EntireName: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation
Components
  • Sample: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Elongation Factor GEF-G
  • RNA: modified formyl-methionine specific initiator transfer RNA
  • RNA: formyl-methionine specific initiator transfer RNA
  • RNA: messenger RNA

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Supramolecule #1000: ribosomal 70S-EF-G complex at an authentic intermediate state dur...

SupramoleculeName: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation
type: sample / ID: 1000 / Number unique components: 5
Molecular weightExperimental: 3 MDa / Theoretical: 3 MDa / Method: sedimentation

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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: K-12
Molecular weightExperimental: 3 MDa / Theoretical: 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 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12

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Macromolecule #2: modified formyl-methionine specific initiator transfer RNA

MacromoleculeName: modified formyl-methionine specific initiator transfer RNA
type: rna / ID: 2 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12

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Macromolecule #3: formyl-methionine specific initiator transfer RNA

MacromoleculeName: formyl-methionine specific initiator transfer RNA / type: rna / ID: 3 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12

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Macromolecule #4: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 4 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12

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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

Concentration0.15 mg/mL
BufferpH: 7.5
Details: 80 mM HEPES potassium, 75 mM NH4Cl, 10 mM MgCl2, 6 mM BME
GridDetails: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 96 K / Instrument: FEI VITROBOT MARK I / Method: blot for 5-10 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder model: GATAN HELIUM
DateJun 14, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.7 µm / Number real images: 308 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each defocus group
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: OTHER / Software - Name: spider / Number images used: 279309

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Atomic model buiding 1

Initial modelPDB ID:

4kix
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4v7b:
Visualization of two tRNAs trapped in transit during EF-G-mediated translocation

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Atomic model buiding 2

Initial modelPDB ID:

4kiy
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4v7b:
Visualization of two tRNAs trapped in transit during EF-G-mediated translocation

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