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- EMDB-6549: Cryo-EM map of EF4-bound ribosomal complexe -

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Basic information

Entry
Database: EMDB / ID: EMD-6549
TitleCryo-EM map of EF4-bound ribosomal complexe
Map data
SampleReconstruction of POST-EF4 complex.:
EF4 / ribosome-prokaryote
Keywordsribosome elongation / GTPase EF4 / tRNA back-translocation / P-loop
Function / homology
Function and homology information


ec:3.6.5.n1: / guanosine tetraphosphate binding / ribosomal large subunit binding / mRNA binding involved in posttranscriptional gene silencing / stringent response / ornithine decarboxylase inhibitor activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcription antitermination factor activity, RNA binding ...ec:3.6.5.n1: / guanosine tetraphosphate binding / ribosomal large subunit binding / mRNA binding involved in posttranscriptional gene silencing / stringent response / ornithine decarboxylase inhibitor activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcription antitermination factor activity, RNA binding / ribosomal small subunit binding / response to salt stress / endoribonuclease inhibitor activity / positive regulation of ribosome biogenesis / negative regulation of endoribonuclease activity / translation repressor activity / transcription antitermination / ribosomal small subunit biogenesis / translation elongation factor activity / negative regulation of translational initiation / four-way junction DNA binding / maturation of LSU-rRNA / mature ribosome assembly / translation repressor activity, mRNA regulatory element binding / polysomal ribosome / positive regulation of RNA splicing / ribosome assembly / assembly of large subunit precursor of preribosome / endodeoxyribonuclease activity / DNA-templated transcription, termination / response to reactive oxygen species / maintenance of translational fidelity / positive regulation of translational fidelity / translational termination / response to cold / regulation of cell growth / positive regulation of translation / ribosomal small subunit assembly / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / small ribosomal subunit rRNA binding / mRNA 5'-UTR binding / response to pH / large ribosomal subunit rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / large ribosomal subunit / ribosomal large subunit assembly / ribosome binding / regulation of translation / 5S rRNA binding / ribosome biogenesis / response to radiation / cytosolic small ribosomal subunit / regulation of mRNA stability / small ribosomal subunit / transferase activity / negative regulation of translation / tRNA binding / ribosome / rRNA binding / structural constituent of ribosome / translation / GTPase activity / mRNA binding / response to antibiotic / GTP binding / negative regulation of transcription, DNA-templated / RNA binding / zinc ion binding / membrane / identical protein binding / plasma membrane / cytosol
Ribosomal protein S17, conserved site / Ribosomal protein L22/L17, conserved site / Ribosomal protein L27, conserved site / Ribosomal protein L33, conserved site / Ribosomal protein L35, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S18, conserved site / Ribosomal protein S21, conserved site ...Ribosomal protein S17, conserved site / Ribosomal protein L22/L17, conserved site / Ribosomal protein L27, conserved site / Ribosomal protein L33, conserved site / Ribosomal protein L35, conserved site / Ribosomal protein S10, conserved site / Ribosomal protein S13, conserved site / Ribosomal protein S14, conserved site / Ribosomal protein S18, conserved site / Ribosomal protein S21, conserved site / Ribosomal protein S3, conserved site / Ribosomal protein L6, bacterial-type / Ribosomal protein L3, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L14P, conserved site / Ribosomal protein S13, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein S9, conserved site / Ribosomal protein L11, conserved site / Ribosomal protein L11, N-terminal / Ribosomal protein L11, C-terminal / Ribosomal protein S7, conserved site / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein S16, conserved site / Ribosomal protein S5 domain 2-type fold / Ribosomal protein S11, bacterial-type / Ribosomal protein L9, N-terminal / Ribosomal protein L9, C-terminal / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25, short-form / Ribosomal protein L6, alpha-beta domain / 30S ribosomal protein S17 / Ribosomal protein L21, conserved site / Ribosomal protein L29, conserved site / Ribosomal protein S6, plastid/chloroplast / Translation protein, beta-barrel domain superfamily / Zinc-binding ribosomal protein / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein S13-like, H2TH / S15/NS1, RNA-binding / Ribosomal protein L9/RNase H1, N-terminal / K homology domain superfamily, prokaryotic type / Translation protein SH3-like domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Ribosomal protein L11, bacterial-type / Elongation factor 4 / Ribosomal protein S12/S23 / Ribosomal protein L30, bacterial-type / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L1, bacterial-type / Nucleic acid-binding, OB-fold / Ribosomal protein L23/L15e core domain superfamily / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein L30, ferredoxin-like fold domain / Ribosomal protein S2, conserved site / Ribosomal S11, conserved site / Ribosomal protein S4, conserved site / Ribosomal protein L30, conserved site / Ribosomal protein L10e/L16 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / K homology domain-like, alpha/beta / 50S ribosomal protein uL4 / Ribosomal protein L2, domain 3 / Ribosomal protein S5 domain 2-type fold, subgroup / Translation elongation factor EF1B/ribosomal protein S6 / GTP-binding protein LepA, C-terminal / Ribosomal protein S5, N-terminal / Ribosomal protein L25/L23 / Ribosomal protein L16, conserved site / Ribosomal protein S6, conserved site / KOW / Ribosomal protein S10 domain superfamily / Ribosomal protein S6 superfamily / Ribosomal protein S8 superfamily / Ribosomal protein L29/L35 superfamily / L21-like superfamily / Ribosomal L18e/L15P superfamily / Ribosomal protein L17 superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein S7 domain superfamily / Ribosomal protein L14 superfamily / Elongation factor 4, domain IV / Ribosomal protein L35 superfamily / Ribosomal protein S14/S29 / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L19 superfamily / Ribosomal protein L33 superfamily / Ribosomal protein S21 superfamily / LepA, C-terminal domain superfamily / Ribosomal protein L28/L24 superfamily / Ribosomal protein S18 superfamily / RNA-binding S4 domain superfamily / Ribosomal protein S11 superfamily / Ribosomal protein L9, N-terminal domain superfamily
30S ribosomal protein S9 / 50S ribosomal protein L13 / 50S ribosomal protein L6 / 50S ribosomal protein L14 / 50S ribosomal protein L16 / 50S ribosomal protein L23 / 30S ribosomal protein S15 / 50S ribosomal protein L17 / 50S ribosomal protein L21 / 30S ribosomal protein S8 ...30S ribosomal protein S9 / 50S ribosomal protein L13 / 50S ribosomal protein L6 / 50S ribosomal protein L14 / 50S ribosomal protein L16 / 50S ribosomal protein L23 / 30S ribosomal protein S15 / 50S ribosomal protein L17 / 50S ribosomal protein L21 / 30S ribosomal protein S8 / 50S ribosomal protein L30 / 50S ribosomal protein L22 / 30S ribosomal protein S14 / 30S ribosomal protein S17 / 50S ribosomal protein L18 / 50S ribosomal protein L2 / 50S ribosomal protein L3 / 50S ribosomal protein L24 / 50S ribosomal protein L4 / Elongation factor 4 / 50S ribosomal protein L5 / 30S ribosomal protein S21 / 30S ribosomal protein S4 / 30S ribosomal protein S5 / 50S ribosomal protein L29 / 30S ribosomal protein S3 / 50S ribosomal protein L33 / 30S ribosomal protein S6 / 30S ribosomal protein S7 / 50S ribosomal protein L15 / 50S ribosomal protein L11 / 50S ribosomal protein L19 / 50S ribosomal protein L1 / 50S ribosomal protein L20 / 50S ribosomal protein L27 / 50S ribosomal protein L28 / 50S ribosomal protein L32 / 50S ribosomal protein L34 / 30S ribosomal protein S2 / 50S ribosomal protein L35 / 50S ribosomal protein L36 / 50S ribosomal protein L9 / 30S ribosomal protein S10 / 30S ribosomal protein S11 / 30S ribosomal protein S12 / 30S ribosomal protein S13 / 30S ribosomal protein S16 / 30S ribosomal protein S18 / 30S ribosomal protein S19 / 30S ribosomal protein S20 / 50S ribosomal protein L25
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang D / Yan K / Liu G / Song G / Luo J / Shi Y / Cheng E / Wu S / Jiang T / Low J ...Zhang D / Yan K / Liu G / Song G / Luo J / Shi Y / Cheng E / Wu S / Jiang T / Low J / Gao N / Qin Y
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome.
Authors: Dejiu Zhang / Kaige Yan / Guangqiao Liu / Guangtao Song / Jiejian Luo / Yi Shi / Erchao Cheng / Shan Wu / Taijiao Jiang / Jizhong Lou / Ning Gao / Yan Qin /
Abstract: EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2- ...EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionNov 30, 2015-
Header (metadata) releaseDec 30, 2015-
Map releaseDec 30, 2015-
UpdateMay 25, 2016-
Current statusMay 25, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcd
  • Surface level: 0.0035
  • 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_6549.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 320 pix.
= 422.4 Å
1.32 Å/pix.
x 320 pix.
= 422.4 Å
1.32 Å/pix.
x 320 pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.0035 / Movie #1: 0.0035
Minimum - Maximum-0.01029744 - 0.02840654
Average (Standard dev.)-0.00002254 (±0.00213855)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0100.028-0.000

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Supplemental data

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Sample components

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Entire Reconstruction of POST-EF4 complex.

EntireName: Reconstruction of POST-EF4 complex. / Number of components: 2

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Component #1: protein, EF4

ProteinName: EF4 / Recombinant expression: Yes
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: ribosome-prokaryote, ribosome 70S

Ribosome-prokaryoteName: ribosome 70S / Prokaryote: ALL / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 20, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 18772
Details: The particles were selected using an automatic selection program.
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143, gold-standard
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Software: COOT / Refinement space: REAL
Details: The agreement between the map and the model was examined in COOT and manually adjusted to obtain the best fit
Input PDB model: 4V9O
Output model

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