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- EMDB-1721: Ribosome dynamics and tRNA movement as visualized by time-resolve... -

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Basic information

Entry
Database: EMDB / ID: 1721
TitleRibosome dynamics and tRNA movement as visualized by time-resolved electron cryomicroscopy
KeywordsRibosome / translation / translocation / tRNA
SampleSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
SourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Synthetic construct
Map dataSubstate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
Methodsingle particle reconstruction, at 12 A resolution
AuthorsFischer N / Konevega AL / Wintermeyer W / Rodnina MV / Stark H
CitationNature, 2010, 466, 329-333

Nature, 2010, 466, 329-333 StrPapers
Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.
Niels Fischer / Andrey L Konevega / Wolfgang Wintermeyer / Marina V Rodnina / Holger Stark

DateDeposition: Apr 30, 2010 / Header (metadata) release: Jun 16, 2010 / Map release: May 6, 2011 / Last update: Apr 30, 2010

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 27
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by height
  • Surface level: 27
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-4v75
  • Surface level: 27
  • Imaged by UCSF CHIMERA
  • Download
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Supplemental images

Downloads & links

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Map

Fileemd_1721.map.gz (map file in CCP4 format, 8193 KB)
Projections & slices
Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
128 pix
2.8 A/pix
= 358.4 A
128 pix
2.8 A/pix
= 358.4 A
128 pix
2.8 A/pix
= 358.4 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 2.8 A
Density
Contour Level:27 (by author), 27 (movie #1):
Minimum - Maximum-122.001 - 202.135
Average (Standard dev.)-1.09276 (20.6321)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions128128128
Origin000
Limit127127127
Spacing128128128
CellA=B=C: 358.4 A
Alpha=beta=gamma: 90 deg.

CCP4 map header:

modeImage stored as Reals
A/pix X/Y/Z2.82.82.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-122.001202.135-1.093

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Supplemental data

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Sample components

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Entire Substate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in class...

EntireName: Substate of E. coli 70S-fMetVal-tRNAVal-tRNAfMet complex in classic post-translocation state (post1)
Number of components: 4
MassTheoretical: 2.5 MDa

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Component #1: ribosome-prokaryote, Ribosome

Ribosome-prokaryoteName: Ribosome / a.k.a: E. coli 70S / Prokaryote: ALL / Recombinant expression: No
MassTheoretical: 2.5 MDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #2: nucleic-acid, fMetVal-tRNAVal

Nucleic-acidName: fMetVal-tRNAVal / a.k.a: peptidyl tRNA / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
MassTheoretical: 25 kDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #3: nucleic-acid, tRNAfMet

Nucleic-acidName: tRNAfMet / a.k.a: deacylated tRNA / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
MassTheoretical: 25 kDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Component #4: nucleic-acid, m022 mRNA

Nucleic-acidName: m022 mRNA / a.k.a: mRNA / Class: RNA / Details: Coding sequence AUGGUU / Structure: SINGLE STRANDED / Synthetic: Yes
SourceSpecies: Synthetic construct

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionBuffer solution: 50 mM Tris-HCl, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 0.6 mM spermine, 0.4 mM spermidine
pH: 7.5
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 77 K / Humidity: 75 % / Method: Manual blotting for about 2 seconds
Time resolved state: Samples were vitrified at different time points along the reaction coordinate (1, 2, 5 and 20 minutes after addition of deacylated tRNAfMet to 70S-fMetVal-tRNAVal complexes)
Details: Vitrification instrument: Custom-built CEVS. Dew-point temperature (temperature on the grid) adjusted to 18 degrees C

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 160 kV / Electron dose: 20 e/A2 / Illumination mode: SPOT SCAN
LensMagnification: 161000 X (nominal), 162740 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 500 - 2000 nm
Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN / Temperature: 77 K
CameraDetector: GENERIC TVIPS (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 13207 / Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Projection matching / Software: IMAGIC, custom, Spider / CTF correction: Local
Details: Final maps were calculated from 13 datasets acquired at different time points, computationally sorted into distinct substates
Resolution: 12 A / Resolution method: FSC 0.5

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Atomic model buiding

Output model

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