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- EMDB-2847: 2.9A structure of E. coli ribosome-EF-Tu complex by Cs-corrected ... -

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Entry
Database: EMDB / ID: EMD-2847
Title2.9A structure of E. coli ribosome-EF-Tu complex by Cs-corrected cryo-EM
Map dataStructure of E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex optimum contour level 0.32-0.64 (1.5-3 sigma)
Sample
  • Sample: E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: elongation factor TuEF-Tu
  • RNA: Phe-tRNAPhe
  • RNA: initiator fMet-tRNAfMet
  • RNA: deacylated tRNAfMet
  • RNA: mRNAMessenger RNA
Keywordsribosome / translation / protein synthesis / decoding / elongation factor Tu / tRNA / RNA modification / antibiotic
Function / homology
Function and homology information


guanyl-nucleotide exchange factor complex / negative regulation of cytoplasmic translational initiation / 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 ...guanyl-nucleotide exchange factor complex / negative regulation of cytoplasmic translational initiation / 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 / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / translation elongation factor activity / translational initiation / negative regulation of DNA-templated DNA replication initiation / 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 / DNA endonuclease activity / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / 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 / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / ribosome biogenesis / regulation of translation / large ribosomal subunit / 5S rRNA binding / cytoplasmic translation / small ribosomal subunit / cytosolic large ribosomal subunit / transferase activity / tRNA binding / negative regulation of translation / molecular adaptor activity / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / GTPase activity / negative regulation of DNA-templated transcription / GTP binding / DNA binding / RNA binding / zinc ion binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : ...Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / 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 L31 type A / 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 L31 signature. / Ribosomal protein S21 / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L10-like domain superfamily / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L11, N-terminal / 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 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/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / 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. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L14P, bacterial-type
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 bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 ...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 bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL31 / 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 / Elongation factor Tu 2 / 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: 2.9 Å
AuthorsFischer N / Neumann P / Konevega AL / Bock LV / Ficner R / Rodnina MV / Stark H
CitationJournal: Nature / Year: 2015
Title: Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM.
Authors: Niels Fischer / Piotr Neumann / Andrey L Konevega / Lars V Bock / Ralf Ficner / Marina V Rodnina / Holger Stark /
Abstract: Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron ...Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.
History
DepositionJan 8, 2015-
Header (metadata) releaseFeb 11, 2015-
Map releaseMar 11, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.6
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  • Surface view with fitted model
  • Atomic models: PDB-5afi
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5afi
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2847.jpg

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Map

FileDownload / File: emd_2847.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex optimum contour level 0.32-0.64 (1.5-3 sigma)
Voxel sizeX=Y=Z: 0.75525 Å
Density
Contour LevelBy EMDB: 0.43 / Movie #1: 0.6
Minimum - Maximum-2.42630196 - 4.7108736
Average (Standard dev.)0.0025036 (±0.21413666)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 317.205 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.755250.755250.75525
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z317.205317.205317.205
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-2.4264.7110.003

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Supplemental data

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Supplemental map: EMD-2847-FSC-mask.map

FileEMD-2847-FSC-mask.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Supplemental map: EMD-2847-full.map

FileEMD-2847-full.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Supplemental map: EMD-2847-half1.map

FileEMD-2847-half1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: EMD-2847-half2.map

FileEMD-2847-half2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfM...

EntireName: E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex
Components
  • Sample: E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: elongation factor TuEF-Tu
  • RNA: Phe-tRNAPhe
  • RNA: initiator fMet-tRNAfMet
  • RNA: deacylated tRNAfMet
  • RNA: mRNAMessenger RNA

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Supramolecule #1000: E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfM...

SupramoleculeName: E. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex
type: sample / ID: 1000 / Number unique components: 6
Molecular weightTheoretical: 2.8 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: elongation factor Tu

MacromoleculeName: elongation factor Tu / type: protein_or_peptide / ID: 1 / Name.synonym: EF-Tu
Details: EF-Tu-GDP stalled on the ribosome by the antibiotic kirromycin
Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: Phe-tRNAPhe

MacromoleculeName: Phe-tRNAPhe / type: rna / ID: 2 / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: initiator fMet-tRNAfMet

MacromoleculeName: initiator fMet-tRNAfMet / type: rna / ID: 3 / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #4: deacylated tRNAfMet

MacromoleculeName: deacylated tRNAfMet / type: rna / ID: 4 / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #5: mRNA

MacromoleculeName: mRNA / type: rna / ID: 5 / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString:
GGCAAGGAGG UAAAUAAUGU UCGUUACGAC

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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

BufferpH: 7.5
Details: 50mM Hepes-KOH, 70mM NH4Cl, 30mM KCl, 20mM MgCl2, 1mM DTT, 0.6mM spermine, 0.4mM spermidine, 0.15mM kirromycin
GridDetails: Quantifoil grids (3.5/1um) covered with pre-floated continuous carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 192000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Using a Cs-corrector from CEOS electron optical aberrations were corrected to residual phase errors of 45degree at scattering angles of >12 to 15 mrad.
DateDec 20, 2011
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Number real images: 24684 / Average electron dose: 40 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: local CTF correction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: OTHER / Software - Name: custom-made, IMAGIC-5, Relion, 1.2 / Number images used: 417201

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