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- EMDB-1553: Pretranslocation ribosome. Group after ML3D classification. Rache... -

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Basic information

Entry
Database: EMDB / ID: EMD-1553
TitlePretranslocation ribosome. Group after ML3D classification. Racheted ribosome with tRNAs in hybrid positions.
Map dataRacheted ribosome with tRNAs in hybrid positions.
Sample
  • Sample: 70 mRNA tRNAfMet fMetLeu-tRNALeu
  • Complex: 70S E.coli ribosome in the pretranslocation state
Keywordsribosome / pretranslocation / hybrid
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 16.0 Å
AuthorsJulian P / Konevega AL / Scheres SHW / Lazaro M / Gil D / Wintermeyer W / Rodnina MV / Valle M
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: Structure of ratcheted ribosomes with tRNAs in hybrid states.
Authors: Patricia Julián / Andrey L Konevega / Sjors H W Scheres / Melisa Lázaro / David Gil / Wolfgang Wintermeyer / Marina V Rodnina / Mikel Valle /
Abstract: During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is ...During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.
History
DepositionSep 8, 2008-
Header (metadata) releaseSep 15, 2008-
Map releaseMay 5, 2009-
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: 32
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 32
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1553.jpg

Downloads & links

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Map

FileDownload / File: emd_1553.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRacheted ribosome with tRNAs in hybrid positions.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 160 pix.
= 352. Å		2.2 Å/pix.
x 160 pix.
= 352. Å		2.2 Å/pix.
x 160 pix.
= 352. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 32.0 / Movie #1: 32
Minimum - Maximum-81.066000000000003 - 192.718999999999994
Average (Standard dev.)3.9672 (±24.584399999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-80-80-80
Dimensions160160160
Spacing160160160
CellA=B=C: 352 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-81-81-81
NX/NY/NZ160160160
MAP C/R/S123
start NC/NR/NS-80-80-80
NC/NR/NS160160160
D min/max/mean-81.066192.7193.967

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Supplemental data

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Sample components

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Entire : 70 mRNA tRNAfMet fMetLeu-tRNALeu

EntireName: 70 mRNA tRNAfMet fMetLeu-tRNALeu
Components
  • Sample: 70 mRNA tRNAfMet fMetLeu-tRNALeu
  • Complex: 70S E.coli ribosome in the pretranslocation state

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Supramolecule #1000: 70 mRNA tRNAfMet fMetLeu-tRNALeu

SupramoleculeName: 70 mRNA tRNAfMet fMetLeu-tRNALeu / type: sample / ID: 1000 / Number unique components: 3

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Supramolecule #1: 70S E.coli ribosome in the pretranslocation state

SupramoleculeName: 70S E.coli ribosome in the pretranslocation state / type: complex / ID: 1 / Name.synonym: pretranslocation ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 50 mM Tris-HCl, pH 7.5 30mM KCl 70 mM NH4Cl 20 mM MgCl2 2 mM spermine
StainingType: NEGATIVE / Details: cryoEM
GridDetails: Quantifoil 300 mesh copper
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: single blot for 1.5 seconds

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Electron microscopy

MicroscopeJEOL 2200FS
TemperatureAverage: 90 K
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Digitization - Sampling interval: 2.2 µm / Average electron dose: 10 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder: single tilt cryoholder / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: volume
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: SPIDER XMIPP / Details: subset after ML3D classification / Number images used: 12590

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