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- PDB-5lkh: Cryo-EM structure of the Tc toxin TcdA1 in its pore state (obtain... -

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Basic information

Entry
Database: PDB / ID: 5lkh
TitleCryo-EM structure of the Tc toxin TcdA1 in its pore state (obtained by flexible fitting)
ComponentsTcdA1
KeywordsTOXIN / nanodisc / injection / pore-forming
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsGatsogiannis, C. / Merino, F. / Prumbaum, D. / Roderer, D. / Leidreiter, F. / Meusch, D. / Raunser, S.
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Membrane insertion of a Tc toxin in near-atomic detail.
Authors: Christos Gatsogiannis / Felipe Merino / Daniel Prumbaum / Daniel Roderer / Franziska Leidreiter / Dominic Meusch / Stefan Raunser /
Abstract: Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual ...Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.
History
DepositionJul 22, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 31, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 7, 2016Group: Database references
Revision 1.2Oct 19, 2016Group: Database references
Revision 1.3Aug 2, 2017Group: Data collection / Experimental preparation / Category: em_sample_support / em_software
Item: _em_sample_support.grid_type / _em_software.name / _em_software.version
Revision 1.4Oct 23, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB

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Assembly

Deposited unit
A: TcdA1
B: TcdA1
C: TcdA1
D: TcdA1
E: TcdA1


Theoretical massNumber of molelcules
Total (without water)1,416,1475
Polymers1,416,1475
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area233500 Å2
MethodPISA

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Components

#1: Protein
TcdA1 / Toxin A


Mass: 283229.406 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: tcdA, tcdA1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9RN43

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Structure of TcdA1 in pore state, determined in lipid nanodiscs
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.4 MDa / Experimental value: NO
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET-19b
Buffer solutionpH: 11
Buffer component
IDConc.FormulaBuffer-ID
120 mMCAPS1
2250 mMNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 15.4 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 1957
Image scansMovie frames/image: 7

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7iMODFITmodel fitting
8DireXmodel fitting
12RELIONinitial Euler assignment
13RELIONfinal Euler assignment
15RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 30061 / Details: particles were picked manually
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 13000
Details: The density was filtered to its local resolution using localfilt (SPARX) and Remap. Although the density map has an average resolution of 3.46 A, the present rough model was obtained by ...Details: The density was filtered to its local resolution using localfilt (SPARX) and Remap. Although the density map has an average resolution of 3.46 A, the present rough model was obtained by flexible fitting within local densities of <9 A resolution.
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Pseudoatomic model was obtained by flexible fitting after initial rigid body fitting of individual domains in Chimera.

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