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- EMDB-1725: Ribosome dynamics and tRNA movement as visualized by time-resolve... -

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Basic information

Entry
Database: EMDB / ID: EMD-1725
TitleRibosome dynamics and tRNA movement as visualized by time-resolved electron cryomicroscopy
Map dataAverage map of E. coli 70S-fMetVal-tRNAVal post-translocation complex prepared at 18 degrees C
Sample
  • Sample: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 18 degrees C
  • Complex: Ribosome
  • RNA: fMetVal-tRNAVal
  • RNA: m022 mRNA
KeywordsRibosome / translation / translocation / tRNA
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 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) releaseMay 13, 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

emd_1725.png

Downloads & links

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Map

FileDownload / File: emd_1725.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAverage map of E. coli 70S-fMetVal-tRNAVal post-translocation complex prepared at 18 degrees C
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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 25.0 / Movie #1: 25
Minimum - Maximum-133.080000000000013 - 189.515999999999991
Average (Standard dev.)-1.41037 (±19.846)
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-133.080189.516-1.410

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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 18 degrees C

EntireName: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 18 degrees C
Components
  • Sample: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 18 degrees C
  • Complex: Ribosome
  • RNA: fMetVal-tRNAVal
  • RNA: m022 mRNA

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Supramolecule #1000: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 18 degrees C

SupramoleculeName: E. coli 70S-fMetVal-tRNAVal post-translocation complex at 18 degrees C
type: sample / ID: 1000 / Number unique components: 3
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: complex / ID: 1 / Name.synonym: E. coli 70S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: fMetVal-tRNAVal

MacromoleculeName: fMetVal-tRNAVal / type: rna / ID: 1 / Name.synonym: peptidyl tRNA / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25 KDa

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Macromolecule #2: m022 mRNA

MacromoleculeName: m022 mRNA / type: rna / ID: 2 / Name.synonym: mRNA / Details: Coding sequence AUGGUU / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 50 mM Tris-HCl, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, 0.6 mM spermine, 0.4 mM spermidine
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Custom-built CEVS. Dew-point temperature (temperature on the grid) adjusted to 18 degrees C
Method: Manual blotting for about 2 seconds

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 77 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 162740 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 161000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Local
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC, custom, Spider / Number images used: 22795

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