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- PDB-9hvc: CryoEM structure of cyclised H-pilus -

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Basic information

Entry
Database: PDB / ID: 9hvc
TitleCryoEM structure of cyclised H-pilus
ComponentsPili assembly chaperone
KeywordsPROTEIN FIBRIL / Pilus / Conjugation / Filament
Function / homologymembrane / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Pili assembly chaperone
Function and homology information
Biological speciesSalmonella (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsIshimoto, N. / Beis, K.
Funding support1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 26, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jul 2, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Pili assembly chaperone
hetero molecules


Theoretical massNumber of molelcules
Total (without water)8,3252
Polymers7,6021
Non-polymers7231
Water55831
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Helical Rise and Twist, mass spectrometry, Cyclisation between N and C terminus.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Pili assembly chaperone


Mass: 7601.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The last five residues of AGIPL are cleaved when H-pilus make cyclisation.
Source: (gene. exp.) Salmonella (bacteria) / Gene: GDL44_23070 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6W0B860
#2: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE


Mass: 722.970 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H75O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 31 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: H-pilus / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 7.1 kDa/nm / Experimental value: YES
Source (natural)Organism: Salmonella (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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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: 1800 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7Buccaneermodel fitting
8UCSF ChimeraXmodel fitting
9Cootmodel fitting
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
14cryoSPARC3D reconstruction
15PHENIX1.20.1_4487model refinement
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 28.93 ° / Axial rise/subunit: 12.14 Å / Axial symmetry: C5
3D reconstructionResolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 123942 / Num. of class averages: 1 / Symmetry type: HELICAL
RefinementHighest resolution: 2.24 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003555
ELECTRON MICROSCOPYf_angle_d0.366738
ELECTRON MICROSCOPYf_dihedral_angle_d14.487105
ELECTRON MICROSCOPYf_chiral_restr0.03581
ELECTRON MICROSCOPYf_plane_restr0.00385

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