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- PDB-9i86: Enterobacteriaphage PRD1 - P12 protein filament in complex with p... -

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Basic information

Entry
Database: PDB / ID: 9i86
TitleEnterobacteriaphage PRD1 - P12 protein filament in complex with poly(dT) ssDNA
Components
  • Single-stranded DNA-binding protein
  • ssDNA poly(dT), 80mer
KeywordsREPLICATION / SSB / Protein primed replication / Protein filament / Homooligomer / ssDNA-binding
Function / homologynucleotide binding / DNA binding / DNA / DNA (> 10) / Single-stranded DNA-binding protein
Function and homology information
Biological speciesEnterobacteria phage PRD1 (virus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsDegen, M. / Traeger, K.L. / Hiller, S.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss Nanoscience Institute Switzerland
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural basis for cooperative ssDNA binding by bacteriophage protein filament P12.
Authors: Lena K Träger / Morris Degen / Joana Pereira / Janani Durairaj / Raphael Dias Teixeira / Sebastian Hiller / Nicolas Huguenin-Dezot /
Abstract: Protein-primed DNA replication is a unique mechanism, bioorthogonal to other known DNA replication modes. It relies on specialised single-stranded DNA (ssDNA)-binding proteins (SSBs) to stabilise ...Protein-primed DNA replication is a unique mechanism, bioorthogonal to other known DNA replication modes. It relies on specialised single-stranded DNA (ssDNA)-binding proteins (SSBs) to stabilise ssDNA intermediates by unknown mechanisms. Here, we present the structural and biochemical characterisation of P12, an SSB from bacteriophage PRD1. High-resolution cryo-electron microscopy reveals that P12 forms a unique, cooperative filament along ssDNA. Each protomer binds the phosphate backbone of 6 nucleotides in a sequence-independent manner, protecting ssDNA from nuclease degradation. Filament formation is driven by an intrinsically disordered C-terminal tail, facilitating cooperative binding. We identify residues essential for ssDNA interaction and link the ssDNA-binding ability of P12 to toxicity in host cells. Bioinformatic analyses place the P12 fold as a distinct branch within the OB-like fold family. This work offers new insights into protein-primed DNA replication and lays a foundation for biotechnological applications.
History
DepositionFeb 4, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Single-stranded DNA-binding protein
Y: ssDNA poly(dT), 80mer
Z: ssDNA poly(dT), 80mer
B: Single-stranded DNA-binding protein
C: Single-stranded DNA-binding protein
D: Single-stranded DNA-binding protein
E: Single-stranded DNA-binding protein
F: Single-stranded DNA-binding protein


Theoretical massNumber of molelcules
Total (without water)148,6078
Polymers148,6078
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Single-stranded DNA-binding protein / Protein P12


Mass: 16671.012 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage PRD1 (virus) / Gene: XII / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P17637
#2: DNA chain ssDNA poly(dT), 80mer


Mass: 24290.438 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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: P12 filament bound to poly(dT) ssDNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Enterobacteria phage PRD1 (virus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: 4200 nm / Nominal defocus min: 390 nm / Cs: 2.7 mm
Image recordingElectron dose: 48.7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2PHENIX1.19.2_4158model refinement
11cryoSPARC4.4.1final Euler assignment
13cryoSPARC4.4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmerty
IDImage processing-IDAngular rotation/subunit (°)Axial rise/subunit (Å)Axial symmetry
1119.18427.461C1
2119.18427.461C1
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 463309 / Symmetry type: HELICAL
RefinementHighest resolution: 2.75 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0077070
ELECTRON MICROSCOPYf_angle_d0.6119714
ELECTRON MICROSCOPYf_dihedral_angle_d15.9772678
ELECTRON MICROSCOPYf_chiral_restr0.0511118
ELECTRON MICROSCOPYf_plane_restr0.0031120

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