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- EMDB-52431: CryoEM structure of cyclised H-pilus -

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Basic information

Entry
Database: EMDB / ID: EMD-52431
TitleCryoEM structure of cyclised H-pilus
Map datasharpened map
Sample
  • Organelle or cellular component: H-pilus
    • Protein or peptide: Pili assembly chaperone
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: water
KeywordsPilus / Conjugation / Filament / PROTEIN FIBRIL
Function / homologymembrane / Pili assembly chaperone
Function and homology information
Biological speciesSalmonella (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsIshimoto N / Beis K
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Cryo-EM structure of the conjugation H-pilus reveals the cyclic nature of the TrhA pilin.
Authors: Naito Ishimoto / Joshua L C Wong / Shan He / Sally Shirran / Olivia Wright-Paramio / Chloe Seddon / Nanki Singh / Carlos Balsalobre / Ravi R Sonani / Abigail Clements / Edward H Egelman / ...Authors: Naito Ishimoto / Joshua L C Wong / Shan He / Sally Shirran / Olivia Wright-Paramio / Chloe Seddon / Nanki Singh / Carlos Balsalobre / Ravi R Sonani / Abigail Clements / Edward H Egelman / Gad Frankel / Konstantinos Beis /
Abstract: Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, ...Conjugation, the major driver of the spread of antimicrobial resistance genes, relies on a conjugation pilus for DNA transfer. Conjugative pili, such as the F-pilus, are dynamic tubular structures, composed of a polymerized pilin, that mediate the initial donor-recipient interactions, a process known as mating pair formation (MPF). IncH are low-copy-number plasmids, traditionally considered broad host range, which are found in bacteria infecting both humans and animals. The reference IncHI1 plasmid R27, isolated from serovar Typhi, encodes the conjugative H-pilus subunit TrhA containing 74 residues after cleavage of the signal sequence. Here, we show that the H-pilus forms long filamentous structures that mediate MPF and describe its cryoelectron-microscopic (cryo-EM) structure at 2.2 Å resolution. Like the F pilus, the H-pilin subunits form helical assemblies with phospholipid molecules at a stoichiometric ratio of 1:1. While there were previous reports that the T-pilus from was composed of cyclic subunits, three recent cryo-EM structures of the T-pilus found no such cyclization. Here, we report that the H-pilin is cyclic, with a covalent bond connecting the peptide backbone between the N and C termini. Both the cryo-EM map and mass spectrometry revealed cleavage of the last five residues of the pilin, followed by cyclization via condensation of the amine and carboxyl residues. Mutagenesis experiments revealed that loss of cyclization abolished pilus biogenesis and efficient plasmid transfer. The cyclic nature of the pilin could stabilize the pilus and may explain the high incidence of IncH plasmid dissemination.
History
DepositionDec 26, 2024-
Header (metadata) releaseMar 26, 2025-
Map releaseMar 26, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52431.png

Downloads & links

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Map

FileDownload / File: emd_52431.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 324 pix.
= 330.48 Å		1.02 Å/pix.
x 324 pix.
= 330.48 Å		1.02 Å/pix.
x 324 pix.
= 330.48 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.02 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.58846295 - 1.151376
Average (Standard dev.)0.0029115144 (±0.054902025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 330.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52431_msk_1.map
Projections & Slices
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Half map: half map A

Fileemd_52431_half_map_1.map
Annotationhalf map A
Projections & Slices
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Half map: half map B

Fileemd_52431_half_map_2.map
Annotationhalf map B
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Sample components

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Entire : H-pilus

EntireName: H-pilus
Components
  • Organelle or cellular component: H-pilus
    • Protein or peptide: Pili assembly chaperone
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: water

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Supramolecule #1: H-pilus

SupramoleculeName: H-pilus / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Salmonella (bacteria)
Molecular weightTheoretical: 7.1 kDa/nm

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Macromolecule #1: Pili assembly chaperone

MacromoleculeName: Pili assembly chaperone / 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 (bacteria)
Molecular weightTheoretical: 7.601941 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSDDGAFGDI WAYMSEALTG APGKIIACGM LFSVAYFGVV KPNLGLALVS ALMMLVMANG EKIISSFLDA GIPL

UniProtKB: Pili assembly chaperone

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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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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 31 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 12.14 Å
Applied symmetry - Helical parameters - Δ&Phi: 28.93 °
Applied symmetry - Helical parameters - Axial symmetry: C5 (5 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 123942
CTF correctionSoftware - Name: cryoSPARC / Type: NONE
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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