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- EMDB-1727: Ribosome dynamics and tRNA movement as visualized by time-resolve... -

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Basic information

Entry
Database: EMDB / ID: EMD-1727
TitleRibosome dynamics and tRNA movement as visualized by time-resolved electron cryomicroscopy
Map data
SampleE. coli 70S-fMetVal-tRNAVal post-translocation complex at 37 degrees C:
ribosome-prokaryote / (nucleic-acidNucleic acid) x 2
KeywordsRibosome / translation / translocation / tRNA
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 18 Å
AuthorsFischer N / Konevega AL / Wintermeyer W / Rodnina MV / Stark H
CitationJournal: Nature / Year: 2010
Title: Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.
Authors: Niels Fischer / Andrey L Konevega / Wolfgang Wintermeyer / Marina V Rodnina / Holger Stark /
Abstract: The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during ...The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.
History
DepositionApr 30, 2010-
Header (metadata) releaseJun 15, 2010-
Map releaseMay 6, 2011-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 25
  • 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_1727.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 128 pix.
= 358.4 Å
2.8 Å/pix.
x 128 pix.
= 358.4 Å
2.8 Å/pix.
x 128 pix.
= 358.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 20.0 / Movie #1: 25
Minimum - Maximum-167.88300000000001 - 254.52000000000001
Average (Standard dev.)-1.38663 (±19.975899999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 358.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/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
α/β/γ90.00090.00090.000
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-167.883254.520-1.387

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Supplemental data

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Sample components

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Entire E. coli 70S-fMetVal-tRNAVal post-translocation complex at 37 degrees C

EntireName: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 37 degrees C
Number of Components: 3
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 (E. coli)

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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 (E. coli)

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Component #3: nucleic-acid, m022 mRNA

nucleic acidName: m022 mRNA / a.k.a: mRNAMessenger RNA / Class: RNA / Details: Coding sequence AUGGUU / Structure: SINGLE STRANDED / Synthetic: Yes
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
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: 95 % / Method: Manual blotting for about 2 seconds
Details: Vitrification instrument: Custom-built CEVS. Dew-point temperature (temperature on the grid) adjusted to 37 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/Å2 / 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
CameraDetector: GENERIC TVIPS (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of Projections: 25455 / Applied Symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Projection matching / Software: IMAGIC, custom, Spider / CTF correction: Local / Resolution: 18 Å / Resolution Method: FSC 0.5

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