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- EMDB-1065: Visualization of release factor 3 on the ribosome during terminat... -

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Basic information

Entry
Database: EMDB / ID: EMD-1065
TitleVisualization of release factor 3 on the ribosome during termination of protein synthesis.
Map dataCryo-EM map of the E.coli 70S/RF3 complex, state 2
Sample
  • Sample: 70S - RF3 complex E.coli
  • Complex: 70S
  • RNA: messenger RNA
  • RNA: isoleucin transfer RNA
  • Protein or peptide: release factor 3
Function / homologyPeptide chain release factor 3
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.5 Å
AuthorsKlaholz BP
CitationJournal: Nature / Year: 2004
Title: Visualization of release factor 3 on the ribosome during termination of protein synthesis.
Authors: Bruno P Klaholz / Alexander G Myasnikov / Marin Van Heel /
Abstract: Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the ...Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In 'state 1', RF3 is pre-bound to the ribosome, whereas in 'state 2' RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.
History
DepositionDec 19, 2003-
Header (metadata) releaseDec 22, 2003-
Map releaseDec 22, 2004-
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: 17.354784071
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 17.354784071
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1065.gif

Downloads & links

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Map

FileDownload / File: emd_1065.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the E.coli 70S/RF3 complex, state 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.42 Å/pix.
x 120 pix.
= 290.28 Å		2.42 Å/pix.
x 120 pix.
= 290.28 Å		2.42 Å/pix.
x 120 pix.
= 290.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.419 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 16.899999999999999 / Movie #1: 17.3547841
Minimum - Maximum-65.829700000000003 - 84.578000000000003
Average (Standard dev.)-0.13018 (±9.802809999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 290.28 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.4192.4192.419
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z290.280290.280290.280
α/β/γ90.00090.00090.000
start NX/NY/NZ0052
NX/NY/NZ12812855
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-65.83084.578-0.130

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Supplemental data

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Sample components

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Entire : 70S - RF3 complex E.coli

EntireName: 70S - RF3 complex E.coli
Components
  • Sample: 70S - RF3 complex E.coli
  • Complex: 70S
  • RNA: messenger RNA
  • RNA: isoleucin transfer RNA
  • Protein or peptide: release factor 3

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Supramolecule #1000: 70S - RF3 complex E.coli

SupramoleculeName: 70S - RF3 complex E.coli / type: sample / ID: 1000
Details: two functional states within a single sample of homogeneous composition, separated by image processing techniques, state 2
Oligomeric state: monomer / Number unique components: 4
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: 70S

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

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Macromolecule #1: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 1 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli
SequenceString:
GGGCCCUUGU UAACAAUUAA GGAGGUAUAC UAUGUUUACG AUUUAAUUGC AGAAAAAAAA AAAAAAAAAA AAA

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Macromolecule #2: isoleucin transfer RNA

MacromoleculeName: isoleucin transfer RNA / type: rna / ID: 2 / Name.synonym: Ile-tRNA / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli

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Macromolecule #3: release factor 3

MacromoleculeName: release factor 3 / type: protein_or_peptide / ID: 3 / Name.synonym: RF3 / Details: PrfC gene / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli / Cell: Escherichia coli
Recombinant expressionOrganism: Escherichia coli MKH13 / Recombinant plasmid: pOSEX3
SequenceInterPro: Peptide chain release factor 3

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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

Concentration0.15 mg/mL
BufferpH: 7.5 / Details: polymix buffer
StainingType: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids
GridDetails: 300 mesh Cu/Rh
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Alignment procedureLegacy - Astigmatism: lens astigmatism was corrected at 60,000 times magnification
DateDec 19, 2001
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 3 µm / Number real images: 44 / Average electron dose: 10 e/Å2 / Details: IMAGE SCIENCE PATCHWORK DENSITOMETER / Bits/pixel: 12
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 48000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: individual particles
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMAGIC / Details: exact filtered back-projection / Number images used: 10011
Final angle assignmentDetails: beta gamma
Final two d classificationNumber classes: 246

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