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- EMDB-1324: Spontaneous reverse movement of mRNA-bound tRNA through the ribosome. -

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Basic information

Entry
Database: EMDB / ID: EMD-1324
TitleSpontaneous reverse movement of mRNA-bound tRNA through the ribosome.
Map datanone
Sample
  • Sample: E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-tRNAVal
  • Complex: E. Coli 70S
  • RNA: fMet-Val-tRNAVal
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsKonevega AL / Fischer N / Semenkov YP / Stark H / Wintermeyer W / Rodnina MV
CitationJournal: Nat Struct Mol Biol / Year: 2007
Title: Spontaneous reverse movement of mRNA-bound tRNA through the ribosome.
Authors: Andrey L Konevega / Niels Fischer / Yuri P Semenkov / Holger Stark / Wolfgang Wintermeyer / Marina V Rodnina /
Abstract: During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, ...During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.
History
DepositionFeb 15, 2007-
Header (metadata) releaseFeb 16, 2007-
Map releaseApr 18, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1324.gif

Downloads & links

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Map

FileDownload / File: emd_1324.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnone
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 Level1: 0.0992 / Movie #1: 0.1
Minimum - Maximum-0.614716 - 0.7856
Average (Standard dev.)-0.000346977 (±0.0781077)
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-72-72-72
NX/NY/NZ144144144
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.6150.786-0.000

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Supplemental data

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Sample components

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Entire : E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-...

EntireName: E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-tRNAVal
Components
  • Sample: E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-tRNAVal
  • Complex: E. Coli 70S
  • RNA: fMet-Val-tRNAVal

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Supramolecule #1000: E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-...

SupramoleculeName: E. Coli 70S-tRNA complex prior to retrotranslocation 70S-fMetVal-tRNAVal
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: E. Coli 70S

SupramoleculeName: E. Coli 70S / type: complex / ID: 1 / Ribosome-details: ribosome-prokaryote: ALL

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Macromolecule #1: fMet-Val-tRNAVal

MacromoleculeName: fMet-Val-tRNAVal / type: rna / ID: 1 / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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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.
GridDetails: 200 mesh copper grid
VitrificationCryogen name: ETHANE / Method: Manual blotting

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Average electron dose: 19 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 166000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsParticles selected semiautomatically.
CTF correctionDetails: local
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: OTHER / Software - Name: Imagic / Number images used: 11732

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