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- EMDB-8416: Time-resolved cryo electron microscopy map of the EF-G-, RRF-, an... -

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Basic information

Entry
Database: EMDB / ID: EMD-8416
TitleTime-resolved cryo electron microscopy map of the EF-G-, RRF-, and tRNA-bound 50S subunit
Map data
SampleRRF, EF-G-, and tRNA-bound 50S subunit:
RRF / tRNATransfer RNA / EF-G
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 16 Å
AuthorsFu Z / Kaledhonkar S / Borg A / Sun M / Chen B / Grassucci RA / Ehrenberg M / Frank J
CitationJournal: Structure / Year: 2016
Title: Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.
Authors: Ziao Fu / Sandip Kaledhonkar / Anneli Borg / Ming Sun / Bo Chen / Robert A Grassucci / Måns Ehrenberg / Joachim Frank /
Abstract: Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination ...Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.
History
DepositionOct 4, 2016-
Header (metadata) releaseOct 19, 2016-
Map releaseNov 23, 2016-
UpdateFeb 14, 2018-
Current statusFeb 14, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8416.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 384 pix.
= 480. Å
1.25 Å/pix.
x 384 pix.
= 480. Å
1.25 Å/pix.
x 384 pix.
= 480. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.013801683 - 0.07032007
Average (Standard dev.)-0.00007234655 (±0.0063684457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 480.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.251.251.25
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z480.000480.000480.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0140.070-0.000

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Supplemental data

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Sample components

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Entire RRF, EF-G-, and tRNA-bound 50S subunit

EntireName: RRF, EF-G-, and tRNA-bound 50S subunit / Number of components: 4

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Component #1: protein, RRF, EF-G-, and tRNA-bound 50S subunit

ProteinName: RRF, EF-G-, and tRNA-bound 50S subunit / Recombinant expression: No

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Component #2: protein, RRF

ProteinName: RRF
Details: His-tagged RRF purified by nickel affinity chromatography.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, tRNA

ProteinName: tRNATransfer RNA / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, EF-G

ProteinName: EF-G / Details: EF-G purified by nickel affinity chromatography / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.7 mg/mL / pH: 7.5
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F30
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.01 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 31000.0 X (nominal) / Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3000.0 nm / Energy filter: None
Specimen HolderModel: SIDE ENTRY, EUCENTRIC / Temperature: (80.0 - K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionSampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 8059
3D reconstructionResolution: 16 Å / Resolution method: FSC 0.143 CUT-OFF

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