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- PDB-9k8v: Cryo-EM structure of the Type II secretion system protein from Vi... -

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Basic information

Entry
Database: PDB / ID: 9k8v
TitleCryo-EM structure of the Type II secretion system protein from Vibrio cholerae
ComponentsGeneral secretion pathway protein D
KeywordsPROTEIN TRANSPORT / Secretion / GspD / nanopore sequencing
Function / homology
Function and homology information


protein secretion by the type II secretion system / type II protein secretion system complex / cell outer membrane / identical protein binding
Similarity search - Function
Type II secretion system protein GspD / : / GspD-like, N0 domain / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / : / NolW-like / NolW-like superfamily / Bacterial type II/III secretion system short domain ...Type II secretion system protein GspD / : / GspD-like, N0 domain / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / : / NolW-like / NolW-like superfamily / Bacterial type II/III secretion system short domain / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
General secretion pathway protein D
Similarity search - Component
Biological speciesVibrio cholerae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsLiu, R.H. / Feng, Q.S. / Zhang, K. / Dai, X. / Dai, J. / Guo, X.R. / Lin, W.F. / Wang, Z.F. / Fu, Y. / Li, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Small / Year: 2025
Title: Tailoring Vibrio-Type Secretin Channel Protein GspD Toward "One-Take" Dual-Constriction Nanopore Sensors.
Authors: Ronghui Liu / Qishun Feng / Kuo Zhang / Xin Dai / Jing Dai / Xinrong Guo / Wenfu Lin / Zhuofei Wang / Qiulan Wu / Yang Fu / Yi Li /
Abstract: Dual-constriction nanopores offer a second sensing region that enhances the interactions with analytes at the single-molecule level. However, existing biological nanopore complexes, i.e., CsgG-CsgF, ...Dual-constriction nanopores offer a second sensing region that enhances the interactions with analytes at the single-molecule level. However, existing biological nanopore complexes, i.e., CsgG-CsgF, are prone to dissociation upon high voltages, enforcing the development of robust "one-take" platforms. Here, Type II general secretin protein D from Vibrio cholerae (VcGspD) as a promising scaffold with dual-constrictions is proposed and engineered. Biochemical analysis reveals that truncation of the N0-N2 domains yields stable multimerization, with the N3 domain being essential. Cryo-electron microscopy (Cryo-EM) resolves the truncated VcGspD (N0-N2) as a 15-mer architecture, confirming its structural integrity and determining localizations of P471 and F472. By introducing a point mutation at position 346 (S346C) and conjugating cholesterol-maleimide, stable channel insertion in lipid bilayers is achieved. Electrophysiological characterization demonstrates a predominantly low-conductance dual-constriction architecture with constriction diameters of ≈2 nm both at the cap and central constriction sites. The F472A mutation, together with the mutations on both constrictions, gives rise to convergent open-channel current and confers high-voltage stability, thus enabling efficient sensing of both single-stranded DNA and polypeptides. The findings establish VcGspD as a promising platform toward dual-constriction nanopore sensing, paving the way for advancements in the development and engineering of secretin channels.
History
DepositionOct 24, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 29, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 29, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.1Nov 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 2.0Dec 24, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / Category: citation / em_admin / Data content type: EM metadata / EM metadata / Item: _citation.journal_volume / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: General secretion pathway protein D
B: General secretion pathway protein D
C: General secretion pathway protein D
D: General secretion pathway protein D
E: General secretion pathway protein D
F: General secretion pathway protein D
G: General secretion pathway protein D
H: General secretion pathway protein D
I: General secretion pathway protein D
J: General secretion pathway protein D
K: General secretion pathway protein D
L: General secretion pathway protein D
M: General secretion pathway protein D
N: General secretion pathway protein D
O: General secretion pathway protein D


Theoretical massNumber of molelcules
Total (without water)683,72015
Polymers683,72015
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
General secretion pathway protein D / Type II secretion system protein GspD / Type II secretion system secretin GspD


Mass: 45581.309 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio cholerae (bacteria)
Gene: gspD, pulD_1, BC353_04215, D6U24_10165, ERS013165_01580, FLM12_02685, QXB71_001789
Production host: Escherichia coli (E. coli) / References: UniProt: A0A085R7B5
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: GspD / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vibrio cholerae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 3000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C15 (15 fold cyclic)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111644 / Symmetry type: POINT

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