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

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Basic information

Entry
Database: EMDB / ID: 5036
TitleAminoacyl-tRNA-EF-Tu-GDP-kir ternary complex-bound E. coli 70S ribosome
Keywordsdecoding / tRNA selection / GTPase / accommodation / flexible fitting / cryo-EM / MDFF / hydrophobic gate / EF-Tu / ribosome / ternary complex
Sample70S ribosome from E. coli complex 70S-fMet-tRNA-Phe-tRNA-EF-Tu-GDP-kirromycin.
SourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Map dataTernary complex-bound 70S E. coli ribosome
Methodsingle particle reconstruction, at 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
CitationProc. Natl. Acad. Sci. U.S.A., 2009, 106, 1063-1068

primary. Proc. Natl. Acad. Sci. U.S.A., 2009, 106, 1063-1068 Yorodumi Papers
Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis.
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. J. Struct. Biol., 2008, 164, 24-32 Yorodumi Papers
Exploration of parameters in cryo-EM leading to an improved density map of the E. coli ribosome.
Jamie LeBarron / Robert A Grassucci / Tanvir R Shaikh / William T Baxter / Jayati Sengupta / Joachim Frank

2. Structure, 2008, 16, 673-683 Yorodumi Papers
Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics.
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
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  • 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
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Supplemental images

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Map

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

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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 package.

Voxel sizeX=Y=Z: 1.2 Å
Density
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 ribosome / Prokaryote: ALL / Recombinant expression: No
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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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 / bacteria / エシェリキア・コリ, 大腸菌 /

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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 / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #4: nucleic-acid, tRNA

Nucleic-acidName: tRNA / a.k.a: deacylated tRNA / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #5: protein, Elongation factor Tu

ProteinName: Elongation factor 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 / bacteria / エシェリキア・コリ, 大腸菌 /
Source (engineered)Expression System: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Source (natural)Location in cell: cytoplasm

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Experimental details

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Sample preparation

Specimen stateparticle
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
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
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
Output model

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