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- EMDB-2711: Cryo-EM map of Trigger Factor bound to a translating ribosome -

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Entry
Database: EMDB / ID: 2711
TitleCryo-EM map of Trigger Factor bound to a translating ribosome
Map dataTnaC-stalled RNC with long nascent chain bound to Trigger Factor (conformation 2)
SampleTnaC stalled RNC with Trigger factor - conformation 2:
ribosome-prokaryote / Trigger factor
Keywordstranslation / co-translational protein folding
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 13.1 Å resolution
AuthorsDeeng J / Chan KY / van der Sluis E / Bischoff L / Berninghausen O / Han W / Gumbart J / Schulten K / Beatrix B / Beckmann R
CitationJournal: J. Mol. Biol. / Year: 2016
Title: Dynamic Behavior of Trigger Factor on the Ribosome.
Authors: J Deeng / K Y Chan / E O van der Sluis / O Berninghausen / W Han / J Gumbart / K Schulten / B Beatrix / R Beckmann
Abstract: Trigger factor (TF) is the only ribosome-associated chaperone in bacteria. It interacts with hydrophobic segments in nascent chain (NCs) as they emerge from the ribosome. TF binds via its N-terminal ...Trigger factor (TF) is the only ribosome-associated chaperone in bacteria. It interacts with hydrophobic segments in nascent chain (NCs) as they emerge from the ribosome. TF binds via its N-terminal ribosome-binding domain (RBD) mainly to ribosomal protein uL23 at the tunnel exit on the large ribosomal subunit. Whereas earlier structural data suggested that TF binds as a rigid molecule to the ribosome, recent comparisons of structural data on substrate-bound, ribosome-bound, and TF in solution from different species suggest that this chaperone is a rather flexible molecule. Here, we present two cryo-electron microscopy structures of TF bound to ribosomes translating an mRNA coding for a known TF substrate from Escherichia coli of a different length. The structures reveal distinct degrees of flexibility for the different TF domains, a conformational rearrangement of the RBD upon ribosome binding, and an increase in rigidity within TF when the NC is extended. Molecular dynamics simulations agree with these data and offer a molecular basis for these observations.
DateDeposition: Jul 14, 2014 / Header (metadata) release: Aug 27, 2014 / Map release: Aug 12, 2015 / Last update: Sep 28, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_2711.map.gz (map file in CCP4 format, 99268 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
294 pix
1.24 Å/pix.
= 363.825 Å
294 pix
1.24 Å/pix.
= 363.825 Å
294 pix
1.24 Å/pix.
= 363.825 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour Level:0.06 (by emdb), 0.06 (movie #1):
Minimum - Maximum-0.35262695 - 0.89136261
Average (Standard dev.)0.01367503 (0.09515595)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderYXZ
Dimensions294294294
Origin-147-147-146
Limit146146147
Spacing294294294
CellA=B=C: 363.82498 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23751.23751.2375
M x/y/z294294294
origin x/y/z0.0000.0000.000
length x/y/z363.825363.825363.825
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S213
start NC/NR/NS-147-147-146
NC/NR/NS294294294
D min/max/mean-0.3530.8910.014

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Supplemental data

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Sample components

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Entire TnaC stalled RNC with Trigger factor - conformation 2

EntireName: TnaC stalled RNC with Trigger factor - conformation 2 / Number of components: 2 / Oligomeric State: monomer

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Component #1: ribosome-prokaryote, cytosolic 70S ribosome

Ribosome-prokaryoteName: cytosolic 70S ribosome / a.k.a: 70S / Prokaryote: ALL / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: KC6

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Component #2: protein, Trigger factor

ProteinName: Trigger factor / Oligomeric Details: 1 / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 48 kDa / Experimental: 48 kDa
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 solutionBuffer solution: 20 mM HEPES, 100 mM KOAc, 10 mM Mg(OAc)2, 2 mM DTT
pH: 7.4
Support filmQuantifoil R3/3 holey carbon supported grids
StainingCryo-EM
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 %
Method: Blot for 10 seconds before plunging, use 2 layers of filter paper V

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jun 4, 2012
Details: Final magnification of the object on the CCD image is 148721
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000 X (nominal), 75000 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1200 - -3600 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 13200 / Sampling size: 15.6 microns / Bit depth: 16

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 72968
Details: The particles were selected using SIGNATURE and processed using SPIDER
3D reconstructionAlgorithm: Projection Matching / Software: SPIDER / CTF correction: on volumes (SPIDER) / Details: Filtered to 13.1 Angstrom / Resolution: 13.1 Å / Resolution method: FSC 0.5, semi-independent

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