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- PDB-30ie: PLD-fold vaccinia virus endonuclease K4 -

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Basic information

Entry
Database: PDB / ID: 30ie
TitlePLD-fold vaccinia virus endonuclease K4
ComponentsVirion nicking-joining enzyme
KeywordsVIRAL PROTEIN / Nuclease / PLD / Phospholipase D / vaccinia virus / poxvirus / DNase
Function / homology
Function and homology information


virion component / endonuclease activity / hydrolase activity
Similarity search - Function
Phospholipase D / PLD-like domain / PLD3/4, PLD-like domain / : / Phospholipase D. Active site motifs. / Phospholipase D/Transphosphatidylase / Phospholipase D phosphodiesterase active site profile.
Similarity search - Domain/homology
Virion nicking-joining enzyme
Similarity search - Component
Biological speciesOrthopoxvirus vaccinia
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsGroger, H. / Burmeister, W.P. / Tarbouriech, N.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-22-CE11-0007-01 France
CitationJournal: J Mol Biol / Year: 2026
Title: High-resolution Structure of the Vaccinia Virus Phospholipase D-fold Endonuclease K4.
Authors: Henri Gröger / Candice Trouba / Jade Barbaste / Guillaume Lacroix / Guy Schoehn / Wim P Burmeister / Nicolas Tarbouriech /
Abstract: Vaccinia virus (VACV) is an orthopoxvirus closely related to mpox virus, which started global outbreaks in 2022. Poxvirus genomes are flanked by short, inverted complementary hairpin telomeres that ...Vaccinia virus (VACV) is an orthopoxvirus closely related to mpox virus, which started global outbreaks in 2022. Poxvirus genomes are flanked by short, inverted complementary hairpin telomeres that feature mismatched bases and insertions essential for viral replication. In this context, a role of the late protein K4 has been proposed. K4 is present in the virion, is apparently non-essential and has a phospholipase D (PLD)-fold as has VACV F13 protein. It also shares fold and nuclease activity with its closest homologue, mammalian PLD3. We established an endonuclease activity against ssDNA and hairpin loops and bubbles in a dsDNA context while RNA is resistant to cleavage. The 2.4 Å cryo-EM structure of K4 shows an unusual octameric assembly, also present in solution. At low concentration, tetramers and dimers similar to the one of hPLD3 are also present. Despite its nuclease activity, in K4 a C-terminal extension blocks the DNA binding pockets. Using an inactive mutant, fortuitously, a DNA 19mer bound simultaneously to 2 sites of the octamer where it displaced the C-termini. DNA binding uses similar residues as the hPLD3 5'-exonuclease, despite different activities and orientations of the DNA. The role of K4 and the control of its activity by the observed auto-inhibition remain enigmatic.
History
DepositionApr 28, 2026Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 19, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 19, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Virion nicking-joining enzyme
H: Virion nicking-joining enzyme
C: Virion nicking-joining enzyme
A: Virion nicking-joining enzyme
F: Virion nicking-joining enzyme
D: Virion nicking-joining enzyme
G: Virion nicking-joining enzyme
E: Virion nicking-joining enzyme


Theoretical massNumber of molelcules
Total (without water)416,7948
Polymers416,7948
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, SAXS, light scattering, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Virion nicking-joining enzyme / Phospholipase-D-like protein K4


Mass: 52099.266 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: RES 1-28 : EXPRESSION TAG Protein fused to N-terminal TEV-cleavable 6xHis-tag
Source: (gene. exp.) Orthopoxvirus vaccinia / Strain: Copenhagen / Gene: OPG042, K4L / Plasmid: pFastBac / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P20537
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Octameric complex of K4 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.392610 MDa / Experimental value: YES
Source (natural)Organism: Vaccinia virus Copenhagen
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Details of virusType: VIRION
Buffer solutionpH: 8 / Details: 20 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: EMS instruments / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3387
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4particle selection
4cryoSPARC4CTF correction
7UCSF ChimeraXmodel fitting
8Cootmodel fitting
10PHENIX2.0_5936model refinement
11cryoSPARC4initial Euler assignment
12cryoSPARC4final Euler assignment
13cryoSPARC4classification
14cryoSPARC43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 754920 / Symmetry type: POINT
Atomic model buildingB value: 97.1 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00427670
ELECTRON MICROSCOPYf_angle_d0.65737630
ELECTRON MICROSCOPYf_dihedral_angle_d4.2293667
ELECTRON MICROSCOPYf_chiral_restr0.054229
ELECTRON MICROSCOPYf_plane_restr0.0054726

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