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

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Basic information

Entry
Database: EMDB / ID: EMD-2847
Title2.9A structure of E. coli ribosome-EF-Tu complex by Cs-corrected cryo-EM
Map data
SampleE. coli 70S-EF-Tu-GDP-kirromycin-Phe-tRNAPhe-fMet-tRNAfmet-tRNAfMet complex:
ribosome-prokaryote / elongation factor TuEF-Tu / (nucleic-acidNucleic acid) x 4
Keywordsribosome / translation / protein synthesis / decoding / elongation factor Tu / tRNA / RNA modification / antibiotic
Function / homology
Function and homology information


translational elongation / stringent response / mRNA binding involved in posttranscriptional gene silencing / ornithine decarboxylase inhibitor activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcription antitermination factor activity, RNA binding / endoribonuclease inhibitor activity / positive regulation of ribosome biogenesis ...translational elongation / stringent response / mRNA binding involved in posttranscriptional gene silencing / ornithine decarboxylase inhibitor activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcription antitermination factor activity, RNA binding / endoribonuclease inhibitor activity / positive regulation of ribosome biogenesis / negative regulation of endoribonuclease activity / translation repressor activity / translation elongation factor activity / transcription antitermination / four-way junction DNA binding / negative regulation of translational initiation / mature ribosome assembly / translation repressor activity, mRNA regulatory element binding / translational initiation / polysomal ribosome / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / ribosome assembly / endodeoxyribonuclease activity / DNA-templated transcription, termination / translational termination / positive regulation of translational fidelity / maintenance of translational fidelity / response to reactive oxygen species / regulation of cell growth / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal small subunit assembly / small ribosomal subunit rRNA binding / mRNA 5'-UTR binding / regulation of mRNA stability / large ribosomal subunit rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / large ribosomal subunit / regulation of translation / ribosome binding / ribosomal large subunit assembly / ribosome biogenesis / response to radiation / 5S rRNA binding / small ribosomal subunit / cytosolic 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 / negative regulation of transcription, DNA-templated / GTP binding / RNA binding / zinc ion binding / membrane / plasma membrane / cytosol
Similarity search - Function
Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu C-terminal domain / Translation elongation factor EFTu/EF1A, C-terminal / Ribosomal protein L10 signature. / Ribosomal protein L10, eubacterial, conserved site / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein L10 / Tr-type G domain, conserved site ...Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu C-terminal domain / Translation elongation factor EFTu/EF1A, C-terminal / Ribosomal protein L10 signature. / Ribosomal protein L10, eubacterial, conserved site / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein L10 / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L31 type A / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein S21 / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein L11, bacterial-type / Ribosomal protein L16 signature 1. / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L17 signature. / Ribosomal protein L10-like domain superfamily / Elongation factor Tu GTP binding domain / Translational (tr)-type GTP-binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L10 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10P / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L28/L24 superfamily / Ribosomal protein L34 signature. / Ribosomal protein L34, conserved site / Ribosomal protein L36 signature. / Ribosomal protein L11, N-terminal / Ribosomal L25p family / Ribosomal protein L11, N-terminal domain / Ribosomal protein L25 / Ribosomal protein L28 / Ribosomal protein L33 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, RNA binding domain / Ribosomal protein L11/L12 / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L35 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L35, conserved site / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L5, bacterial-type / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L18, bacterial-type / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L20 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L6, bacterial-type / Ribosomal protein S2 signature 2. / Ribosomal protein L36 / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L14P, bacterial-type / Ribosomal protein S20 / Ribosomal protein S19, bacterial-type / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein L2, bacterial/organellar-type / 30S ribosomal protein S17
Similarity search - Domain/homology
50S ribosomal protein L16 / 50S ribosomal protein L17 / 30S ribosomal protein S15 / 50S ribosomal protein L23 / 30S ribosomal protein S5 / 50S ribosomal protein L14 / 50S ribosomal protein L13 / 30S ribosomal protein S9 / 30S ribosomal protein S8 / 50S ribosomal protein L30 ...50S ribosomal protein L16 / 50S ribosomal protein L17 / 30S ribosomal protein S15 / 50S ribosomal protein L23 / 30S ribosomal protein S5 / 50S ribosomal protein L14 / 50S ribosomal protein L13 / 30S ribosomal protein S9 / 30S ribosomal protein S8 / 50S ribosomal protein L30 / 50S ribosomal protein L21 / Elongation factor Tu 2 / 50S ribosomal protein L6 / 30S ribosomal protein S14 / 30S ribosomal protein S17 / 50S ribosomal protein L18 / 30S ribosomal protein S3 / 50S ribosomal protein L2 / 50S ribosomal protein L3 / 50S ribosomal protein L24 / 50S ribosomal protein L4 / 50S ribosomal protein L22 / 50S ribosomal protein L5 / 30S ribosomal protein S21 / 30S ribosomal protein S4 / 30S ribosomal protein S13 / 30S ribosomal protein S2 / 50S ribosomal protein L31 / 30S ribosomal protein S6 / 30S ribosomal protein S7 / 50S ribosomal protein L15 / 50S ribosomal protein L10 / 50S ribosomal protein L11 / 50S ribosomal protein L19 / 50S ribosomal protein L20 / 50S ribosomal protein L27 / 50S ribosomal protein L28 / 50S ribosomal protein L29 / 50S ribosomal protein L32 / 30S ribosomal protein S20 / 50S ribosomal protein L33 / 50S ribosomal protein L34 / 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 S16 / 30S ribosomal protein S18 / 30S ribosomal protein S19 / 50S ribosomal protein L25
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
  • 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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  • 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

Downloads & links

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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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.76 Å/pix.
x 420 pix.
= 317.205 Å
0.76 Å/pix.
x 420 pix.
= 317.205 Å
0.76 Å/pix.
x 420 pix.
= 317.205 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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

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Supplemental map: EMD-2847-full.map

FileEMD-2847-full.map
Projections & Slices
AxesZYX

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Supplemental map: EMD-2847-half1.map

FileEMD-2847-half1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Supplemental map: EMD-2847-half2.map

FileEMD-2847-half2.map
Projections & Slices
AxesZYX

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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
Number of Components: 6
MassTheoretical: 2.8 MDa

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Component #1: ribosome-prokaryote, 70S ribosome

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

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Component #2: protein, elongation factor Tu

ProteinName: elongation factor TuEF-Tu / a.k.a: EF-Tu
Details: EF-Tu-GDP stalled on the ribosome by the antibiotic kirromycin
Recombinant expression: Yes
SourceSpecies: Escherichia coli (E. coli)

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Component #3: nucleic-acid, Phe-tRNAPhe

nucleic acidName: Phe-tRNAPhe / Class: T-RNA / Structure: OTHER / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #4: nucleic-acid, initiator fMet-tRNAfMet

nucleic acidName: initiator fMet-tRNAfMet / Class: T-RNA / Structure: OTHER / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #5: nucleic-acid, deacylated tRNAfMet

nucleic acidName: deacylated tRNAfMet / Class: T-RNA / Structure: OTHER / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #6: nucleic-acid, mRNA

nucleic acidName: mRNAMessenger RNA / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: Yes
Sequence:
GGCAAGGAGG UAAAUAAUGU UCGUUACGAC
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionBuffer solution: 50mM Hepes-KOH, 70mM NH4Cl, 30mM KCl, 20mM MgCl2, 1mM DTT, 0.6mM spermine, 0.4mM spermidine, 0.15mM kirromycin
pH: 7.5
Support filmQuantifoil grids (3.5/1um) covered with pre-floated continuous carbon
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: Dec 20, 2011
Electron gunElectron Source: OTHER / Accelerating Voltage: 300 kV / Electron Dose: 40 e/Å2 / Illumination Mode: SPOT SCAN
LensMagnification: 192000 X (nominal)
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.
Cs: 0.01 mm / Imaging Mode: BRIGHT FIELD / Defocus: 700 - 2500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON I (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 24684

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 417201
3D reconstructionSoftware: custom-made, IMAGIC-5, Relion, 1.2 / CTF correction: local CTF correction / Resolution: 2.9 Å / Resolution Method: FSC 0.143, gold-standard

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Atomic model buiding

Output model

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