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- EMDB-2696: Cryo-EM map of Trigger Factor bound to a translating ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-2696
TitleCryo-EM map of Trigger Factor bound to a translating ribosome
Map dataTnaC-stalled RNC with long nascent chain bound to Trigger Factor (conformation1)
Sample
  • Sample: TnaC stalled RNC with Trigger factor - conformation 1
  • Complex: cytosolic 70S ribosome
  • Protein or peptide: Trigger factor
Keywordstranslation / co-translational protein folding
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 13.1 Å
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.
History
DepositionJun 27, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseJul 15, 2015-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.2
  • 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_2696.map.gz / Format: CCP4 / Size: 94.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTnaC-stalled RNC with long nascent chain bound to Trigger Factor (conformation1)
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy EMDB: 0.8 / Movie #1: 0.2
Minimum - Maximum-0.58389419 - 1.59712231
Average (Standard dev.)0.02163768 (±0.1833843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-147-147-146
Dimensions294294294
Spacing294294294
CellA=B=C: 363.82498 Å
α=β=γ: 90.0 °

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-0.5841.5970.022

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Supplemental data

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Sample components

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Entire : TnaC stalled RNC with Trigger factor - conformation 1

EntireName: TnaC stalled RNC with Trigger factor - conformation 1
Components
  • Sample: TnaC stalled RNC with Trigger factor - conformation 1
  • Complex: cytosolic 70S ribosome
  • Protein or peptide: Trigger factor

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Supramolecule #1000: TnaC stalled RNC with Trigger factor - conformation 1

SupramoleculeName: TnaC stalled RNC with Trigger factor - conformation 1 / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2

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Supramolecule #1: cytosolic 70S ribosome

SupramoleculeName: cytosolic 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: KC6

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Macromolecule #1: Trigger factor

MacromoleculeName: Trigger factor / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 48 KDa / Theoretical: 48 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

BufferpH: 7.4 / Details: 20 mM HEPES, 100 mM KOAc, 10 mM Mg(OAc)2, 2 mM DTT
StainingType: NEGATIVE / Details: Cryo-EM
GridDetails: Quantifoil R3/3 holey carbon supported grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III
Method: Blot for 10 seconds before plunging, use 2 layers of filter paper V

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 75000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.6 µm / Nominal defocus min: -1.2 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsFinal magnification of the object on the CCD image is 148721
DateJun 4, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 13200 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: on volumes (SPIDER)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.1 Å / Resolution method: OTHER / Software - Name: SPIDER / Details: Filtered to 13.1 Angstrom / Number images used: 100931
DetailsThe particles were selected using SIGNATURE and processed using SPIDER

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