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- EMDB-65024: CryoEM structure of phospholipid-independent cyclised RP4 pilus -

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Basic information

Entry
Database: EMDB / ID: EMD-65024
TitleCryoEM structure of phospholipid-independent cyclised RP4 pilus
Map datasharpened map
Sample
  • Organelle or cellular component: P-pilus
    • Protein or peptide: TrbC/VIRB2 family protein
KeywordsPilus / Conjugation / Antibiotic / PROTEIN FIBRIL
Function / homologyConjugal transfer TrbC/type IV secretion VirB2 / TrbC/VIRB2 pilin / TrbC/VIRB2 family protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsIshimoto N / Beis K
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2026
Title: Phospholipid-independent biogenesis and function of the RP4 conjugation pilus.
Authors: Naito Ishimoto / Shan He / Mikhail Bogdanov / Terry K Smith / Gad Frankel / Konstantinos Beis /
Abstract: Bacterial conjugation, the process of horizontal gene transfer between bacteria, is initiated by mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated ...Bacterial conjugation, the process of horizontal gene transfer between bacteria, is initiated by mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.74 Å resolution. Uniquely, both the structural and quantitative mass spectral analyses revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of F- (pKpQIL), H- (R27) or W- (R388) pili, whose biogenesis and DNA transfer is PG-dependent. RP4 is the first example of a lipid-independent functional mating pilus. This discovery suggests that an amphipathic lipid moiety is not universally essential for the biogenesis of conjugative pili and MPF, providing an alternative model for their assembly and function. These data expand our understanding of the diverse bacterial mechanisms employ to transfer genetic material.
History
DepositionJun 10, 2025-
Header (metadata) releaseMay 27, 2026-
Map releaseMay 27, 2026-
UpdateJul 1, 2026-
Current statusJul 1, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_65024.png

Downloads & links

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Map

FileDownload / File: emd_65024.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.2 Å/pix.
x 196 pix.
= 235.2 Å		1.2 Å/pix.
x 196 pix.
= 235.2 Å		1.2 Å/pix.
x 196 pix.
= 235.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065
Minimum - Maximum-0.13348532 - 0.3068865
Average (Standard dev.)0.0023082865 (±0.019482369)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 235.20001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_65024_msk_1.map
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Additional map: map

Fileemd_65024_additional_1.map
Annotationmap
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Half map: half map 1

Fileemd_65024_half_map_1.map
Annotationhalf map 1
Projections & Slices
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Histogram

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Half map: half map 2

Fileemd_65024_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : P-pilus

EntireName: P-pilus
Components
  • Organelle or cellular component: P-pilus
    • Protein or peptide: TrbC/VIRB2 family protein

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Supramolecule #1: P-pilus

SupramoleculeName: P-pilus / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.14 kDa/nm

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Macromolecule #1: TrbC/VIRB2 family protein

MacromoleculeName: TrbC/VIRB2 family protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.139474 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SEGTGGSLPY ESWLTNLRNS VTGPVAFALS IIGIVVAGGV LIFGGELNAF FRTLIFLVLV MALLVGAQNV MSTFFGRG

UniProtKB: TrbC/VIRB2 family protein

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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
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) / Number grids imaged: 1 / Number real images: 3405 / 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 / 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
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: 15.15 Å
Applied symmetry - Helical parameters - Δ&Phi: 24.62 °
Applied symmetry - Helical parameters - Axial symmetry: C5 (5 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 10674
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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