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- EMDB-64923: Cryo-EM structure of PLD3 bound to ssDNA (poly(A)) -

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Basic information

Entry
Database: EMDB / ID: EMD-64923
TitleCryo-EM structure of PLD3 bound to ssDNA (poly(A))
Map data
Sample
  • Complex: PLD3 bound to poly(A) substrate
    • Complex: PLD3
      • Protein or peptide: 5'-3' exonuclease PLD3
    • Complex: ssDNA
      • DNA: DNA (5'-D(*AP*AP*AP*AP*A)-3')
Keywordsexonuclease / IMMUNE SYSTEM
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
5'-3' exonuclease PLD3
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsHirano Y / Ezaki W / Ohto U / Shimizu T
Funding support Japan, 3 items
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, 2025-
Header (metadata) releaseDec 24, 2025-
Map releaseDec 24, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_64923.png

Downloads & links

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Map

FileDownload / File: emd_64923.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å		0.83 Å/pix.
x 300 pix.
= 249. Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.632804 - 1.15786
Average (Standard dev.)0.0000422398 (±0.029477492)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64923_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64923_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : PLD3 bound to poly(A) substrate

EntireName: PLD3 bound to poly(A) substrate
Components
  • Complex: PLD3 bound to poly(A) substrate
    • Complex: PLD3
      • Protein or peptide: 5'-3' exonuclease PLD3
    • Complex: ssDNA
      • DNA: DNA (5'-D(*AP*AP*AP*AP*A)-3')

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Supramolecule #1: PLD3 bound to poly(A) substrate

SupramoleculeName: PLD3 bound to poly(A) substrate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: PLD3

SupramoleculeName: PLD3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others) / Synthetically produced: Yes

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Macromolecule #1: DNA (5'-D(*AP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(*AP*AP*AP*AP*A)-3') / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.30217 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)

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Macromolecule #2: 5'-3' exonuclease PLD3

MacromoleculeName: 5'-3' exonuclease PLD3 / type: protein_or_peptide / ID: 2
Details: C-terminal 6 residues are derived from the expression tag.
Number of copies: 2 / Enantiomer: LEVO / EC number: spleen exonuclease
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.467531 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EYGDLHLFGP NQRPAPCYDP CEAVLVESIP EGLDFPNAST GNPSTSQAWL GLLAGAHSSL DIASFYWTLT NNDTHTQEPS AQQGEEVLR QLQTLAPKGV NVRIAVSKPS GPQPQADLQA LLQSGAQVRM VDMQKLTHGV LATKFWVVDQ THFYLGSANM D WRSLTQVK ...String:
EYGDLHLFGP NQRPAPCYDP CEAVLVESIP EGLDFPNAST GNPSTSQAWL GLLAGAHSSL DIASFYWTLT NNDTHTQEPS AQQGEEVLR QLQTLAPKGV NVRIAVSKPS GPQPQADLQA LLQSGAQVRM VDMQKLTHGV LATKFWVVDQ THFYLGSANM D WRSLTQVK ELGVVMYNCS CLARDLTKIF EAYWFLGQAG SSIPSTWPRF YDTRYNQETP MEICLNGTPA LAYLASAPPP LC PSGRTPD LKALLNVVDN ARSFIYVAVM NYLPTLEFSH PHRFWPAIDD GLRRATYERG VKVRLLISCW GHSEPSMRAF LLS LAALRD NHTHSDIQVK LFVVPADEAQ ARIPYARVNA NKYMVTERAT YIGTSNWSGN YFTETAGTSL LVTQNGRGGL RSQL EAIFL RDWDSPYSHD LDTSADSVGN ACRLLLEVLF Q

UniProtKB: 5'-3' exonuclease PLD3

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 5.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 711544
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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