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- EMDB-2552: Electron cryo-microscopy of TcdA1 in pore state. -

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Basic information

Entry
Database: EMDB / ID: EMD-2552
TitleElectron cryo-microscopy of TcdA1 in pore state.
Map dataReconstruction of TcdA1 in pore state
Sample
  • Sample: TcdA1
  • Protein or peptide: TcdA1
KeywordsPhotorhabdus / Tc toxin / ABC toxin / translocation
Function / homology
Function and homology information


identical protein binding
Similarity search - Function
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
Similarity search - Domain/homology
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: 0.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2552.png

Downloads & links

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Map

FileDownload / File: emd_2552.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of TcdA1 in pore state
Voxel sizeX=Y=Z: 2.5 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-1.22805309 - 3.91189599
Average (Standard dev.)0.01138122 (±0.11420764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-110-110-110
Dimensions220220220
Spacing220220220
CellA=B=C: 550.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z550.000550.000550.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-110-110-110
NC/NR/NS220220220
D min/max/mean-1.2283.9120.011

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Supplemental data

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Sample components

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Entire : TcdA1

EntireName: TcdA1
Components
  • Sample: TcdA1
  • Protein or peptide: TcdA1

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Supramolecule #1000: TcdA1

SupramoleculeName: TcdA1 / type: sample / ID: 1000 / Oligomeric state: Pentamer / Number unique components: 1
Molecular weightExperimental: 1.41 MDa / Theoretical: 1.41 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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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: 11
Details: 50 mM CAPS,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: 0.0029 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
DateAug 27, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 605 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Each Particle
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: Sparx / Number images used: 13303

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Atomic model buiding 1

Initial modelPDB ID:

4o9y

SoftwareName: DireX
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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