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- PDB-9vbh: Cryo-EM structure of human PLD3 bound to ssDNA (poly(T)) -

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Basic information

Entry
Database: PDB / ID: 9vbh
TitleCryo-EM structure of human PLD3 bound to ssDNA (poly(T))
Components
  • 5'-3' exonuclease PLD3
  • DNA (5'-D(*TP*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 / myotube differentiation / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / D-type glycerophospholipase activity / regulation of cytokine production involved in inflammatory response / immune system process / Role of phospholipids in phagocytosis / lysosomal lumen ...spleen exonuclease / Synthesis of PG / single-stranded DNA 5'-3' DNA exonuclease activity / myotube differentiation / Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases / D-type glycerophospholipase activity / regulation of cytokine production involved in inflammatory response / immune system process / Role of phospholipids in phagocytosis / lysosomal lumen / lipid metabolic process / late endosome membrane / early endosome membrane / inflammatory response / Golgi membrane / lysosomal membrane / endoplasmic reticulum membrane / extracellular exosome
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
DNA / DNA (> 10) / 5'-3' exonuclease PLD3
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 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 PLD3
B: DNA (5'-D(*TP*TP*TP*T)-3')
C: 5'-3' exonuclease PLD3
D: DNA (5'-D(*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,34812
Polymers126,6564
Non-polymers4,6928
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 PLD3 / (S / S)-bis(monoacylglycero)phosphate synthase PLD3 / HindIII K4L homolog / Hu-K4 / Phospholipase D3


Mass: 48467.531 Da / Num. of mol.: 2 / Mutation: H201A/H416A
Source method: isolated from a genetically manipulated source
Details: C-terminal 6 residues are derived from the expression tag.
Source: (gene. exp.) Homo sapiens (human) / Gene: PLD3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: Q8IV08, spleen exonuclease, Hydrolases; Acting on ester bonds; Phosphoric-diester hydrolases
#2: DNA chain DNA (5'-D(*TP*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.: 4
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: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
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
1Homodimer of PLD3 luminal domainCOMPLEX#1-#20RECOMBINANT
2Homodimer of the luminal domain of PLD3COMPLEX#11RECOMBINANT
3ssDNACOMPLEX#21SYNTHETIC
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23synthetic 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: 800 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: C2 (2 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 510357 / Symmetry type: POINT
RefinementCross valid method: NONE

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