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- EMDB-1370: Progression of the ribosome recycling factor through the ribosome... -

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Basic information

Entry
Database: EMDB / ID: EMD-1370
TitleProgression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits.
Map data70S-RRF complex
Sample
  • Sample: 70S_RRF_UG
  • Complex: 70S Escherichia coli ribosome
  • Ligand: Ribosome Recycling Factor
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.3 Å
AuthorsBarat C / Datta PP / Raj VS / Sharma MR / Kaji H / Kaji A / Agrawal RK
CitationJournal: Mol Cell / Year: 2007
Title: Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits.
Authors: Chandana Barat / Partha P Datta / V Samuel Raj / Manjuli R Sharma / Hideko Kaji / Akira Kaji / Rajendra K Agrawal /
Abstract: After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling ...After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.
History
DepositionMay 31, 2007-
Header (metadata) releaseJun 1, 2007-
Map releaseJan 7, 2008-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.000279618
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.000279618
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_1370.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation70S-RRF complex
Voxel sizeX=Y=Z: 2.76 Å
Density
Contour Level1: 0.000369 / Movie #1: 0.0002796
Minimum - Maximum-0.000555101 - 0.00130594
Average (Standard dev.)0.0000202832 (±0.000150449)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 358.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.762.762.76
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z358.800358.800358.800
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/NS130130130
D min/max/mean-0.0010.0010.000

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Supplemental data

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Sample components

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Entire : 70S_RRF_UG

EntireName: 70S_RRF_UG
Components
  • Sample: 70S_RRF_UG
  • Complex: 70S Escherichia coli ribosome
  • Ligand: Ribosome Recycling Factor

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Supramolecule #1000: 70S_RRF_UG

SupramoleculeName: 70S_RRF_UG / type: sample / ID: 1000 / Number unique components: 2
Molecular weightTheoretical: 2.5205 MDa

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Supramolecule #1: 70S Escherichia coli ribosome

SupramoleculeName: 70S Escherichia coli ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.6 MDa

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Macromolecule #1: Ribosome Recycling Factor

MacromoleculeName: Ribosome Recycling Factor / type: ligand / ID: 1 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 20.5 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: 8.2mM MgSO4, 80mM NH4Cl, 10mM Tris (pH 7.4), without DTT
GridDetails: Quantifoil 300 mesh Cu grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Wadsworth Centers fabricated guillotine plunger
Method: 5ul of specimen was applied to the grid, then blotted using Whatman number 1 filter paper for 2 to 4 seconds, then plunged.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50760 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Oxford cryo-transfer holder / Specimen holder model: OTHER
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism corrected and 210,000X magnification.
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 109 / Average electron dose: 20 e/Å2 / Bits/pixel: 12
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 15.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 36439

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Atomic model buiding 1

SoftwareName: O
DetailsProtocol: Rigid Body
RefinementProtocol: RIGID BODY FIT

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