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- EMDB-62181: Cryo-EM structure of the Type II secretion system protein from Vi... -

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Basic information

Entry
Database: EMDB / ID: EMD-62181
TitleCryo-EM structure of the Type II secretion system protein from Vibrio cholerae
Map data
Sample
  • Complex: GspD
    • Protein or peptide: General secretion pathway protein D
KeywordsSecretion / GspD / nanopore sequencing / PROTEIN TRANSPORT
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsLiu RH / Feng QS / Zhang K / Dai X / Dai J / Guo XR / Lin WF / Wang ZF / Fu Y / Li Y
Funding support1 items
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, 2024-
Header (metadata) releaseOct 29, 2025-
Map releaseOct 29, 2025-
UpdateNov 19, 2025-
Current statusNov 19, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62181.png

Downloads & links

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Map

FileDownload / File: emd_62181.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.67 Å/pix.
x 480 pix.
= 320.64 Å		0.67 Å/pix.
x 480 pix.
= 320.64 Å		0.67 Å/pix.
x 480 pix.
= 320.64 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.668 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum-13.517094999999999 - 27.792169999999999
Average (Standard dev.)0.000000000002428 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 320.63998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62181_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_62181_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : GspD

EntireName: GspD
Components
  • Complex: GspD
    • Protein or peptide: General secretion pathway protein D

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Supramolecule #1: GspD

SupramoleculeName: GspD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: General secretion pathway protein D

MacromoleculeName: General secretion pathway protein D / type: protein_or_peptide / ID: 1 / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 45.581309 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGNNRVVYLK YAKAEDLVEV LKGVSENLQA EKGTGQPTTS KRNEVMIAAH ADTNSLVLTA PQDIMNAMLE VIGQLDIRRA QVLIEALIV EMAEGDGINL GVQWGSLESG SVIQYGNTGA SIGNVMIGLE EAKDTTQTKA VYDTNNNFLR NETTTTKGDY T KLASALSS ...String:
MGNNRVVYLK YAKAEDLVEV LKGVSENLQA EKGTGQPTTS KRNEVMIAAH ADTNSLVLTA PQDIMNAMLE VIGQLDIRRA QVLIEALIV EMAEGDGINL GVQWGSLESG SVIQYGNTGA SIGNVMIGLE EAKDTTQTKA VYDTNNNFLR NETTTTKGDY T KLASALSS IQGAAVSIAM GDWTALINAV SNDSSSNILS SPSITVMDNG EASFIVGEEV PVITGSTAGS NNDNPFQTVD RK EVGIKLK VVPQINEGNS VQLNIEQEVS NVLGANGAVD VRFAKRQLNT SVMVQDGQML VLGGLIDERA LESESKVPLL GDI PLLGQL FRSTSSQVEK KNLMVFIKPT IIRDGVTADG ITQRKYNYIR AEQLFRAEKG LRLLDDASVP VLPKFGDDRR HSPE IQAFI EQMEAKQWSH PQFEK

UniProtKB: General secretion pathway protein D

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C15 (15 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 111644
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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