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- EMDB-5036: Aminoacyl-tRNA-EF-Tu-GDP-kir ternary complex-bound E. coli 70S ri... -

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Entry
Database: EMDB / ID: 5036
TitleAminoacyl-tRNA-EF-Tu-GDP-kir ternary complex-bound E. coli 70S ribosome
Map dataTernary complex-bound 70S E. coli ribosome
Sample70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.Ribosome:
ribosome-prokaryote / (nucleic-acidNucleic acid) x 3 / Elongation factor TuEF-Tu
Keywordsdecoding / tRNA selection / GTPase / accommodation / flexible fitting / cryo-EM / MDFF / hydrophobic gate / EF-Tu / ribosome / ternary complex
Function / homologyRibosomal protein S9, bacterial/plastid / Ribosomal protein L25 / Ribosomal protein L36 superfamily / Ribosomal protein L20, C-terminal / L28p-like / Elongation factor Tu, domain 2 / Ribosomal protein L5, N-terminal / Ribosomal protein L5, C-terminal / Tr-type G domain, conserved site / Ribosomal protein L15 ...Ribosomal protein S9, bacterial/plastid / Ribosomal protein L25 / Ribosomal protein L36 superfamily / Ribosomal protein L20, C-terminal / L28p-like / Elongation factor Tu, domain 2 / Ribosomal protein L5, N-terminal / Ribosomal protein L5, C-terminal / Tr-type G domain, conserved site / Ribosomal protein L15 / Ribosomal protein L21-like / Ribosomal protein S8 superfamily / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein S10 domain / 30s ribosomal protein S13, C-terminal / P-loop containing nucleoside triphosphate hydrolase / Ribosomal protein L28/L24 / Ribosomal protein S16 domain superfamily / Ribosomal protein S7 domain / Ribosomal protein L1-like / Ribosomal protein S6 superfamily / Ribosomal protein L29/L35 superfamily / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S7 domain superfamily / Ribosomal protein S11 superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L10e/L16 superfamily / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L13 superfamily / Ribosomal protein S18 superfamily / Ribosomal protein L14 superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / L21-like superfamily / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein S20 superfamily / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein L22/L17 superfamily / Ribosomal protein L17 superfamily / Ribosomal L18e/L15P superfamily / Ribosomal protein L1, conserved site / Ribosomal protein S19, superfamily / Ribosomal protein L28/L24 superfamily / Ribosomal protein S11, bacterial-type / Ribosomal protein S9, conserved site / Ribosomal protein S5 domain 2-type fold / 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 S13, bacterial-type / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein S17, conserved site / Ribosomal protein L14P, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L3, conserved site / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, conserved site / Ribosomal protein S21, conserved site / Ribosomal protein S18, conserved site / Ribosomal protein S16, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein L4 domain superfamily / Ribosomal protein L18e/L15P / Ribosomal protein L13, conserved site / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L5 domain superfamily / Ribosomal protein S4/S9 / Ribosomal protein L2, conserved site / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L35 / Ribosomal protein L34, conserved site / Ribosomal protein L11, C-terminal / Ribosomal protein S19 conserved site / Ribosomal protein L5, bacterial-type / Ribosomal protein L5, conserved site / Ribosomal protein S6, conserved site / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L16, conserved site / Ribosomal protein L11, conserved site / Ribosomal protein L11, N-terminal / RNA-binding S4 domain superfamily / Ribosomal protein L35 superfamily / Ribosomal protein S13, conserved site / Ribosomal protein S20 / KH domain / Ribosomal protein L9, C-terminal domain / Ribosomal Proteins L2, C-terminal domain / Ribosomal protein L11, N-terminal domain / Ribosomal protein S5, C-terminal domain / Elongation factor Tu domain 2 / Elongation factor Tu C-terminal domain / Ribosomal L32p protein family / Ribosomal protein L35 / Ribosomal protein L14 signature.
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 6.7 Å resolution
AuthorsVilla E / Sengupta J / Trabuco LG / LeBarron J / Baxter WT / Shaikh TR / Grassucci RA / Nissen P / Ehrenberg M / Schulten K / Frank J
Citation
Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2009
Title: Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis.
Authors: Elizabeth Villa / Jayati Sengupta / Leonardo G Trabuco / Jamie LeBarron / William T Baxter / Tanvir R Shaikh / Robert A Grassucci / Poul Nissen / Måns Ehrenberg / Klaus Schulten / Joachim Frank
#1: Journal: J. Struct. Biol. / Year: 2008
Title: Exploration of parameters in cryo-EM leading to an improved density map of the E. coli ribosome.
Authors: Jamie LeBarron / Robert A Grassucci / Tanvir R Shaikh / William T Baxter / Jayati Sengupta / Joachim Frank
#2: Journal: Structure / Year: 2008
Title: Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics.
Authors: Leonardo G Trabuco / Elizabeth Villa / Kakoli Mitra / Joachim Frank / Klaus Schulten
Validation ReportPDB-ID: 4v69

SummaryFull reportAbout validation report
DateDeposition: Dec 1, 2008 / Header (metadata) release: Feb 25, 2009 / Map release: May 5, 2009 / Last update: Jul 23, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 80
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 80
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-4v69
  • Surface level: 80
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5036_1.png

Downloads & links

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Map

Fileemd_5036.map.gz (map file in CCP4 format, 115250 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
309 pix
1.2 Å/pix.
= 370.8 Å		309 pix
1.2 Å/pix.
= 370.8 Å		309 pix
1.2 Å/pix.
= 370.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density

Histogram

Histogram (log scale)
Contour Level:90 (by author), 80 (movie #1):
Minimum - Maximum-114.57819366 - 303.00439453
Average (Standard dev.)6.03115606 (29.14512062)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions309309309
Origin-155-155-155
Limit153153153
Spacing309309309
CellA=B=C: 370.80002 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z309309309
origin x/y/z0.0000.0000.000
length x/y/z370.800370.800370.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS-155-155-155
NC/NR/NS309309309
D min/max/mean-114.578303.0046.031

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Supplemental data

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Sample components

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Entire 70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GD...

EntireName: 70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
Number of components: 5 / Oligomeric State: single particle

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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: nucleic-acid, fMet-tRNAfMet

Nucleic-acidName: fMet-tRNAfMet / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #3: nucleic-acid, Phe-tRNAPhe

Nucleic-acidName: Phe-tRNAPhe / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #4: nucleic-acid, tRNA

Nucleic-acidName: tRNATransfer RNA / a.k.a: deacylated tRNA / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #5: protein, Elongation factor Tu

ProteinName: Elongation factor TuEF-Tu / a.k.a: EF-Tu / Oligomeric Details: monomer / Details: EF-Tu bound to kirromycin and GDP / Number of Copies: 1 / Recombinant expression: Yes
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)
Source (natural)Location in cell: cytoplasm

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.0768 mg/ml
Buffer solution: 5 mM potassium phosphate, 5 mM magnesium acetate, 5 mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8 mM putrescine, 1 mM spermidine, and 1 mM dithioerythritol
pH: 7.5
Support filmThin Carbon on 300 mesh Quantifoil R2/4
VitrificationInstrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 80 K / Humidity: 90 % / Method: Blot 3 seconds before plunging
Details: Vitrification instrument: FEI vitrobot. Blot Offset at -1 mm

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300 / Date: Aug 31, 2006 / Details: Low dose
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 58279 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 115,000 times magnification with fastscan ccd
Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 4520 nm
Specimen HolderHolder: FEI Polara Cartridge / Model: OTHER / Temperature: 84 K
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 304 / Scanner: ZEISS SCAI / Sampling size: 7 microns / Bit depth: 12 / OD range: 1.2

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 131599 / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: weighted back projection / Software: Spider
CTF correction: Correction of reconstruction of each defocus group
Resolution: 6.7 Å
Resolution method: FCS at 0.5 cut-off criterion, extrapolated to full dataset

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Atomic model buiding

Modeling #1Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
Input PDB model: 2I2U

2i2u

Modeling #2Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
Input PDB model: 2I2V

2i2v

Modeling #3Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
Details: Protocol: MDFF. An atomic model of the entire ribosome and factors was creating using molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
Input PDB model: 1OB2

1ob2

Output model

4v69

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