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- EMDB-2551: Electron cryo-microscopy of the PTC3 holotoxin complex (TcdA1, Tc... -

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Basic information

Entry
Database: EMDB / ID: EMD-2551
TitleElectron cryo-microscopy of the PTC3 holotoxin complex (TcdA1, TcdB2, TccC3) in prepore state
Map dataPTC3 Holotoxin Complex
Sample
  • Sample: PTC3 (TcdA1, TcdB2, TccC3)
  • Protein or peptide: TcdA1
  • Protein or peptide: TccC3
  • Protein or peptide: TcdB2
KeywordsPhotorabdus / Tc toxin / ABC toxin / cargo-delivery
Function / homology
Function and homology information


extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region ...Insecticide toxin TcdB middle/C-terminal / Insecticide toxin TcdB middle/N-terminal / Insecticide toxin TcdB middle/C-terminal region / Insecticide toxin TcdB middle/N-terminal region / Salmonella virulence plasmid 65kDa B protein / Salmonella virulence plasmid 65kDa B protein / Toxin complex C-like repeat / Tripartite Tc toxins repeat / ABC toxin, N-terminal domain / ABC toxin N-terminal region / TcA receptor binding domain / TcA receptor binding domain / Insecticidal toxin complex/plasmid virulence protein / Tc toxin complex TcA, C-terminal TcB-binding domain / Neuraminidase-like domain / Salmonella virulence plasmid 28.1kDa A protein / Tc toxin complex TcA C-terminal TcB-binding domain / Neuraminidase-like domain / Rhs repeat-associated core / Integrin alpha, N-terminal
Similarity search - Domain/homology
Insecticidal toxin complex protein TcdB2 / Insecticidal toxin complex protein TccC3 / TcdA1
Similarity search - Component
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsGatsogiannis C / Meusch D / Efremov RG / Lang AE / Hofnagel O / Vetter IR / Aktories K / Raunser S
CitationJournal: Nature / Year: 2014
Title: Mechanism of Tc toxin action revealed in molecular detail.
Authors: Dominic Meusch / Christos Gatsogiannis / Rouslan G Efremov / Alexander E Lang / Oliver Hofnagel / Ingrid R Vetter / Klaus Aktories / Stefan Raunser /
Abstract: Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly ...Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed β-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.
History
DepositionJan 7, 2014-
Header (metadata) releaseFeb 5, 2014-
Map releaseMar 12, 2014-
UpdateApr 9, 2014-
Current statusApr 9, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.31
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.31
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2551.png

Downloads & links

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Map

FileDownload / File: emd_2551.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPTC3 Holotoxin Complex
Voxel sizeX=Y=Z: 1.25 Å
Density
Contour LevelBy AUTHOR: 0.31 / Movie #1: 0.31
Minimum - Maximum-0.56139374 - 2.12666035
Average (Standard dev.)0.00626538 (±0.06813836)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 640.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.251.251.25
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z640.000640.000640.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-0.5612.1270.006

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Supplemental data

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Sample components

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Entire : PTC3 (TcdA1, TcdB2, TccC3)

EntireName: PTC3 (TcdA1, TcdB2, TccC3)
Components
  • Sample: PTC3 (TcdA1, TcdB2, TccC3)
  • Protein or peptide: TcdA1
  • Protein or peptide: TccC3
  • Protein or peptide: TcdB2

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Supramolecule #1000: PTC3 (TcdA1, TcdB2, TccC3)

SupramoleculeName: PTC3 (TcdA1, TcdB2, TccC3) / type: sample / ID: 1000
Oligomeric state: TcdB2/TccC3 bind to one homopentamer of TcdA1
Number unique components: 3
Molecular weightExperimental: 1.68 MDa / Theoretical: 1.68 MDa

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Macromolecule #1: TcdA1

MacromoleculeName: TcdA1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: Pentamer / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightExperimental: 283 KDa / Theoretical: 283 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: TcdA1

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Macromolecule #2: TccC3

MacromoleculeName: TccC3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightTheoretical: 109 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Insecticidal toxin complex protein TccC3

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Macromolecule #3: TcdB2

MacromoleculeName: TcdB2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Molecular weightExperimental: 166 KDa / Theoretical: 166 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Insecticidal toxin complex protein TcdB2

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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

Concentration0.02 mg/mL
BufferpH: 5
Details: 50 mM MES, 100 mM NaCl, 0.05% Tween-20, 5% glycerol
GridDetails: C-Flat 2/1-4C copper 400 mesh, with additional thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 1 sec before plunging

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 124472 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 0.0032 µm / Nominal defocus min: 0.0004 µm / Nominal magnification: 60000
Specialist opticsEnergy filter - Name: in-column Omega filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 15.0 eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
DateNov 19, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 1250 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: Sparx
Details: TcdA1 component of the complex has an average resolution of 7.4 A, whereas the TcdB2/TccC3 components an average resolution of 11 A. Local resolution calculations were performed using a 20 A ...Details: TcdA1 component of the complex has an average resolution of 7.4 A, whereas the TcdB2/TccC3 components an average resolution of 11 A. Local resolution calculations were performed using a 20 A sphere for windowed-FSC calculations at 0.5.
Number images used: 43000
DetailsPartial-symmetry projection matching approach (after each refinement round, C5 symmetry was applied for the TcdA1 component).

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Atomic model buiding 1

Initial modelPDB ID:

4o9x

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4o9y

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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