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- PDB-9vbk: Cryo-EM structure of human PLD4 bound to ssDNA (poly(T)) -

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Basic information

Entry
Database: PDB / ID: 9vbk
TitleCryo-EM structure of human PLD4 bound to ssDNA (poly(T))
Components
  • 5'-3' exonuclease PLD4
  • DNA (5'-D(*TP*TP*T)-3')
KeywordsIMMUNE SYSTEM / Exonuclease
Function / homology
Function and homology information


spleen exonuclease / Synthesis of PG / single-stranded DNA 5'-3' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / regulation of cytokine production involved in inflammatory response / Synthesis of IP3 and IP4 in the cytosol / Role of phospholipids in phagocytosis / phagocytosis / hematopoietic progenitor cell differentiation / phagocytic vesicle ...spleen exonuclease / Synthesis of PG / single-stranded DNA 5'-3' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / regulation of cytokine production involved in inflammatory response / Synthesis of IP3 and IP4 in the cytosol / Role of phospholipids in phagocytosis / phagocytosis / hematopoietic progenitor cell differentiation / phagocytic vesicle / trans-Golgi network membrane / establishment of localization in cell / lipid metabolic process / early endosome / lysosome / inflammatory response / innate immune response / endoplasmic reticulum membrane / endoplasmic reticulum / nucleus
Similarity search - Function
PLD-like domain / PLD-like domain / : / Phospholipase D. Active site motifs. / Phospholipase D/Transphosphatidylase / Phospholipase D phosphodiesterase active site profile.
Similarity search - Domain/homology
THYMIDINE-3'-PHOSPHATE / DNA / DNA (> 10) / 5'-3' exonuclease PLD4
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsHirano, Y. / Ezaki, W. / Ohto, U. / Shimizu, T.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22K06110 Japan
Japan Society for the Promotion of Science (JSPS)25K09523 Japan
Japan Science and TechnologyJPMJCR21E4 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Mechanistic insights into single-stranded DNA degradation by lysosomal exonucleases PLD3 and PLD4 from structural snapshots.
Authors: Yoshinori Hirano / Wakiko Ezaki / Ryota Sato / Umeharu Ohto / Kensuke Miyake / Toshiyuki Shimizu /
Abstract: Lysosomal exonuclease phospholipase D (PLD) family PLD3 and PLD4 degrade single-stranded RNA or DNA and regulate TLR7 or TLR9 responses. Polymorphisms of these enzymes are associated with human ...Lysosomal exonuclease phospholipase D (PLD) family PLD3 and PLD4 degrade single-stranded RNA or DNA and regulate TLR7 or TLR9 responses. Polymorphisms of these enzymes are associated with human diseases: PLD4 is associated with inflammatory diseases, and PLD3 is associated with neurodegenerative diseases. Here, we determine the structures of substrate-bound PLD3 and PLD4 by cryo-electron microscopy. Our structures reveal that PLD3 rebuilds a substrate-binding pocket, depending on the substrate, mainly via motion of the Phe335-containing loop. Furthermore, we captured the structure in a metastable state that appears during substrate rearrangement following product release. Together, our findings identify the residues that underlie the distinct activities of PLD3 and PLD4. This study provides a mechanistic basis for the exonuclease activity of PLD3 and PLD4 in single-stranded DNA degradation.
History
DepositionJun 4, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 24, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data 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
A: 5'-3' exonuclease PLD4
B: 5'-3' exonuclease PLD4
C: 5'-3' exonuclease PLD4
D: 5'-3' exonuclease PLD4
F: DNA (5'-D(*TP*TP*T)-3')
G: DNA (5'-D(*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)221,90630
Polymers214,8706
Non-polymers7,03624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
5'-3' exonuclease PLD4 / (S / S)-bis(monoacylglycero)phosphate synthase PLD4 / Phospholipase D family member 4 / Phospholipase D4


Mass: 46287.273 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PLD4, C14orf175, UNQ2488/PRO5775 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: Q96BZ4, spleen exonuclease, Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases
#2: DNA chain DNA (5'-D(*TP*TP*T)-3')


Mass: 14860.490 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Chemical ChemComp-T3P / THYMIDINE-3'-PHOSPHATE / ALPHA-ANOMERIC THYMIDINE-3'-PHOSPHATE


Type: DNA linking / Mass: 322.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N2O8P
Has ligand of interestN
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

Component
IDNameTypeEntity IDParent-IDSource
1Homo-tetramer of PLD4 luminal domainCOMPLEX#1-#20MULTIPLE SOURCES
2Homodimer of the luminal domain of PLD3COMPLEX#11RECOMBINANT
3ssDNACOMPLEX#21SYNTHETIC
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33synthetic construct (others)32630
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 5.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65742 / Symmetry type: POINT
RefinementCross valid method: NONE

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