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- PDB-8cq2: Photorhabdus luminescens TcdA1 prepore-to-pore intermediate, C16S... -

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Basic information

Entry
Database: PDB / ID: 8cq2
TitlePhotorhabdus luminescens TcdA1 prepore-to-pore intermediate, C16S, C20S, C870S, T1279C mutant
ComponentsTcdA1
KeywordsTOXIN / Bacterial toxin / Tc toxin
Function / homology
Function and homology information


identical protein binding
Similarity search - Function
: / TcdA1, receptor binding domain / : / TcdA1, receptor binding domain / 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 ...: / TcdA1, receptor binding domain / : / TcdA1, receptor binding domain / 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: 3.6 Å
AuthorsNganga, P.N. / Roderer, D. / Belyy, A. / Prumbaum, D. / Raunser, S.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Sci Adv / Year: 2025
Title: Multistate kinetics of the syringe-like injection mechanism of Tc toxins.
Authors: Peter Njenga Ng'ang'a / Julian Folz / Svetlana Kucher / Daniel Roderer / Ying Xu / Oleg Sitsel / Alexander Belyy / Daniel Prumbaum / Ralf Kühnemuth / Tufa E Assafa / Min Dong / Claus A M ...Authors: Peter Njenga Ng'ang'a / Julian Folz / Svetlana Kucher / Daniel Roderer / Ying Xu / Oleg Sitsel / Alexander Belyy / Daniel Prumbaum / Ralf Kühnemuth / Tufa E Assafa / Min Dong / Claus A M Seidel / Enrica Bordignon / Stefan Raunser /
Abstract: Tc toxins are pore-forming virulence factors of many pathogenic bacteria. Following pH-induced conformational changes, they perforate the target membrane like a syringe to translocate toxic enzymes ...Tc toxins are pore-forming virulence factors of many pathogenic bacteria. Following pH-induced conformational changes, they perforate the target membrane like a syringe to translocate toxic enzymes into a cell. Although this complex transformation has been structurally well studied, the reaction pathway and the resulting temporal evolution have remained elusive. We used an integrated biophysical approach to monitor prepore-to-pore transition and found a reaction time of ~30 hours for a complete transition. We show two asynchronous general steps of the process, shell opening and channel ejection, with the overall reaction pathway being a slow multistep process involving three intermediates. Liposomes, an increasingly high pH, or receptors facilitate shell opening, which is directly correlated with an increased rate of the prepore-to-pore transition. Channel ejection is a near-instantaneous process which occurs with a transition time of <60 milliseconds. Understanding the mechanism of action of Tc toxins and unveiling modulators of the kinetics are key steps toward their application as biomedical devices or biopesticides.
History
DepositionMar 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)1,426,6485
Polymers1,426,6485
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
TcdA1 / Toxin A


Mass: 285329.625 Da / Num. of mol.: 5 / Mutation: C16S C20S C870S T1279C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Photorhabdus luminescens (bacteria) / Gene: tcdA, tcdA1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 RIPL / References: UniProt: Q9RN43
Has protein modificationN

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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: TcdA1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.5 MDa / Experimental value: YES
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 RIPL / Plasmid: pET19
Buffer solutionpH: 11.2
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMCAPSC9H19NO3S1
2150 mMSodium ChlorideNaCl1
30.1 %Tween-201
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 78 e/Å2 / Detector mode: INTEGRATING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13059

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10SPHIREinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 573814
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80710 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00986195
ELECTRON MICROSCOPYf_angle_d0.89117070
ELECTRON MICROSCOPYf_dihedral_angle_d13.66931490
ELECTRON MICROSCOPYf_chiral_restr0.05312980
ELECTRON MICROSCOPYf_plane_restr0.00715205

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