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- EMDB-65235: CryoEM structure of cyclised H-pilus (D69N) -

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Basic information

Entry
Database: EMDB / ID: EMD-65235
TitleCryoEM structure of cyclised H-pilus (D69N)
Map datasharpening map
Sample
  • Organelle or cellular component: H-Pilus (D69N)
    • Protein or peptide: Pilin
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
Keywordspilus / conjugation / filament / PROTEIN FIBRIL
Function / homologyPilin
Function and homology information
Biological speciesSalmonella enterica subsp. enterica serovar Typhi (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsIshimoto N / Beis K
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2026
Title: H pilin cyclisation and pilus biogenesis are promiscuous but electrostatic perturbations impair conjugation efficiency.
Authors: Shan He / Naito Ishimoto / Joshua L C Wong / Sophia David / Julia Sanchez-Garrido / Mikhail Bogdanov / Konstantinos Beis / Gad Frankel /
Abstract: During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 ...During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg does not prevent cyclisation or pilus formation, which remains structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlates with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolish conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membrane's inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation is rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.
History
DepositionJul 2, 2025-
Header (metadata) releaseDec 24, 2025-
Map releaseDec 24, 2025-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_65235.png

Downloads & links

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Map

FileDownload / File: emd_65235.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpening map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 256 pix.
= 305.152 Å		1.19 Å/pix.
x 256 pix.
= 305.152 Å		1.19 Å/pix.
x 256 pix.
= 305.152 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.192 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.3
Minimum - Maximum-4.2567797 - 7.459442
Average (Standard dev.)0.011344927 (±0.39201605)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 305.152 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_65235_msk_1.map
Projections & Slices
AxesZYX

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Additional map: map

Fileemd_65235_additional_1.map
Annotationmap
Projections & Slices
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Half map: half map 1

Fileemd_65235_half_map_1.map
Annotationhalf map 1
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_65235_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : H-Pilus (D69N)

EntireName: H-Pilus (D69N)
Components
  • Organelle or cellular component: H-Pilus (D69N)
    • Protein or peptide: Pilin
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

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Supramolecule #1: H-Pilus (D69N)

SupramoleculeName: H-Pilus (D69N) / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi (bacteria)
Molecular weightTheoretical: 7.1 kDa/nm

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Macromolecule #1: Pilin

MacromoleculeName: Pilin / type: protein_or_peptide / ID: 1
Details: The last five residues of AGIPL are cleaved when H-pilus make cyclisation.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhi (bacteria)
Molecular weightTheoretical: 7.600957 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSDDGAFGDI WAYMSEALTG APGKIIACGM LFSVAYFGVV KPNLGLALVS ALMMLVMANG EKIISSFLNA GIPL

UniProtKB: Pilin

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Macromolecule #2: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 2 / Number of copies: 1 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 12.24 Å
Applied symmetry - Helical parameters - Δ&Phi: 29.00 °
Applied symmetry - Helical parameters - Axial symmetry: C5 (5 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1477240
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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