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- EMDB-1077: Visualization of ribosome-recycling factor on the Escherichia col... -

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Basic information

Entry
Database: EMDB / ID: EMD-1077
TitleVisualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications.
Map dataComplex of e.coli 70S ribosome and ribosome recycling factor (RRF).
Sample
  • Sample: 70S-RRF complex of e.coli
  • Complex: 70S
  • Protein or peptide: RRF
Function / homologyRibosome recycling factor / Ribosome recycling factor domain / RRF superfamily / Ribosome recycling factor / translational termination / cytoplasm / Ribosome-recycling factor
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsAgrawal RK
CitationJournal: Proc Natl Acad Sci U S A / Year: 2004
Title: Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: functional implications.
Authors: Rajendra K Agrawal / Manjuli R Sharma / Michael C Kiel / Go Hirokawa / Timothy M Booth / Christian M T Spahn / Robert A Grassucci / Akira Kaji / Joachim Frank /
Abstract: After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosome-recycling factor (RRF), together with elongation factor G (EF-G), ...After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosome-recycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by approximately 13 A) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA.
History
DepositionApr 14, 2004-
Header (metadata) releaseApr 14, 2004-
Map releaseApr 14, 2005-
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: 0.0012
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1t1m, PDB-1t1o
  • Surface level: 0.0012
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1t1m
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1t1o
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1077.gif

Downloads & links

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Map

FileDownload / File: emd_1077.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex of e.coli 70S ribosome and ribosome recycling factor (RRF).
Voxel sizeX=Y=Z: 2.82 Å
Density
Contour Level1: 0.0017 / Movie #1: 0.0012
Minimum - Maximum-0.00396945 - 0.00668052
Average (Standard dev.)0.000286843 (±0.000751109)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z366.600366.600366.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-0.0040.0070.000

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Supplemental data

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Sample components

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Entire : 70S-RRF complex of e.coli

EntireName: 70S-RRF complex of e.coli
Components
  • Sample: 70S-RRF complex of e.coli
  • Complex: 70S
  • Protein or peptide: RRF

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Supramolecule #1000: 70S-RRF complex of e.coli

SupramoleculeName: 70S-RRF complex of e.coli / type: sample / ID: 1000 / Number unique components: 2
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa / Method: sedimentation

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Supramolecule #1: 70S

SupramoleculeName: 70S / type: complex / ID: 1 / Ribosome-details: ribosome-prokaryote: ALL
Molecular weightExperimental: 2.48 MDa / Theoretical: 2.48 MDa

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Macromolecule #1: RRF

MacromoleculeName: RRF / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: e.coli / synonym: bacteria / Cell: bacteria / Location in cell: cytosol
Molecular weightExperimental: 20 KDa / Theoretical: 20 KDa

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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: 52 mM Tris-HCL (pH7.5), 25 mM KCL, 5.5 mM NH4Cl, 11 mM Mg(OAc)2 0.3 mM Beta-ME
GridDetails: quantifoil grids with holy carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: two side blotting plunger
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49696 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.4 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Cryo transfer / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
DateJun 1, 2002
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 76 / Average electron dose: 20 e/Å2 / Od range: 1.2 / Bits/pixel: 12
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups
Final angle assignmentDetails: SPIDER: theta 15 degrees, phi 15 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Final map was calculated from 23 CTF corrected defocus groups
Number images used: 51217

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Atomic model buiding 1

SoftwareName: manual
DetailsProtocol: Rigid Body. the RRF was fitted as single rigid body
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation coefficient
Output model

PDB-1t1m:
Binding position of ribosome recycling factor (RRF) on the E. coli 70S ribosome

PDB-1t1o:
Components of the control 70S ribosome to provide reference for the RRF binding site

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