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- PDB-9ots: Cryo-EM structure of the T9SS PORkN ring complex of P. Gingivalis -

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Basic information

Entry
Database: PDB / ID: 9ots
TitleCryo-EM structure of the T9SS PORkN ring complex of P. Gingivalis
Components
  • Por secretion system protein porK/gldK
  • Por secretion system protein porN/gldN
KeywordsMEMBRANE PROTEIN / T9SS / Type IX SECRETION SYSTEM / PORKN RING COMPLEX / PORPHYROMONAS GINGIVALIS / PROTEIN TRANSPORT
Function / homology
Function and homology information


formylglycine-generating oxidase activity
Similarity search - Function
Gliding motility associated protein GldN / Gliding motility associated protein GldN / : / Sulfatase-modifying factor enzyme / Sulfatase-modifying factor enzyme 1-like domain / Sulfatase-modifying factor enzyme superfamily / C-type lectin fold / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Por secretion system protein porN/gldN / Por secretion system protein porK/gldK
Similarity search - Component
Biological speciesPorphyromonas gingivalis ATCC 33277 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu, X. / Song, L. / Zheng, L. / Hu, B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: mBio / Year: 2025
Title: Structure of the T9SS PorKN ring complex reveals conformational plasticity based on the repurposed FGE fold.
Authors: Xiangan Liu / John D Perpich / Liqiang Song / Christian Cambillau / Thierry Doan / Lei Zheng / Richard J Lamont / Eric Cascales / Bo Hu /
Abstract: The type IX secretion system (T9SS) is a protein secretion machinery unique to the Bacteroidetes-Chlorobi-Fibrobacteres superphylum, which plays crucial roles in bacterial pathogenesis and gliding ...The type IX secretion system (T9SS) is a protein secretion machinery unique to the Bacteroidetes-Chlorobi-Fibrobacteres superphylum, which plays crucial roles in bacterial pathogenesis and gliding motility. It is composed of >15 proteins, including the proton-motive force-dependent PorLM motor, the PorKN ring anchored to the outer membrane, and the Sov translocon. Here, we present the cryo-electron microscopy (EM) structure of the PorKN ring complex from at 3.2 Å resolution. Our structural analysis reveals that PorK contains a repurposed formylglycine-generating enzyme-like fold, which serves as a structural hub for complex assembly rather than enzymatic activity. The complex exhibits a 33-fold symmetry with PorK and PorN assembling two tightly packed and wedged subrings. The structure reveals previously uncharacterized N- and C-terminal helices in PorN that are crucial for PorK binding and complex stability. By combining our high-resolution structure with cryo-electron tomography data, we propose a mechanism whereby PorKN undergoes conformational changes during substrate transport, transitioning between 50° and 90° states relative to the membrane plane. Finally, structural predictions coupled to site-directed disulfide cross-linking identified contacts between PorM and the PorKN ring. Collectively, these findings provide crucial insights into the molecular architecture and dynamic behavior of the T9SS machinery, advancing our understanding of bacterial protein secretion mechanisms.IMPORTANCEThe bacterial type IX secretion system (T9SS) is essential for processes such as gliding motility and secretion of virulence factors. In , a major periodontal pathogen, the T9SS transports over 30 virulence-associated proteins, making it central to disease development. The T9SS core is composed of PorLM motors that are thought to energize the PorKN outer membrane-associated ring. However, the molecular architecture of the PorKN ring has remained unresolved. Here, we present its atomic-resolution cryo-EM structure, revealing a formylglycine-generating enzyme-like fold in PorK that mediates PorK-PorN interactions through specific insertion motifs. Our results show that the ring exhibits intrinsic structural plasticity, including dynamic flexibility and variable stoichiometry. AlphaFold models and disulfide cross-linking experiments further provide information on how PorLM motors are connected to the PorKN ring. These insights redefine our understanding of the T9SS mechanism of action and offer a structural framework for the development of targeted antimicrobial strategies.
History
DepositionMay 27, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 20, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Sep 24, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Por secretion system protein porK/gldK
B: Por secretion system protein porK/gldK
C: Por secretion system protein porK/gldK
D: Por secretion system protein porN/gldN
E: Por secretion system protein porN/gldN
F: Por secretion system protein porN/gldN


Theoretical massNumber of molelcules
Total (without water)292,4966
Polymers292,4966
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Por secretion system protein porK/gldK


Mass: 55979.594 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Porphyromonas gingivalis ATCC 33277 (bacteria)
Strain: ATCC 33277 / References: UniProt: B2RLF0
#2: Protein Por secretion system protein porN/gldN


Mass: 41519.016 Da / Num. of mol.: 3 / Source method: isolated from a natural source
Source: (natural) Porphyromonas gingivalis ATCC 33277 (bacteria)
Strain: ATCC 33277 / References: UniProt: B2RLE7
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: T9SS PorKN ring complex / Type: ORGANELLE OR CELLULAR COMPONENT / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Porphyromonas gingivalis ATCC 33277 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 5000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2500000 / Symmetry type: POINT
RefinementHighest resolution: 3.2 Å

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