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- EMDB-64925: Cryo-EM structure of human PLD4 bound to ssDNA (poly(T)) -

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Basic information

Entry
Database: EMDB / ID: EMD-64925
TitleCryo-EM structure of human PLD4 bound to ssDNA (poly(T))
Map data
Sample
  • Complex: Homo-tetramer of PLD4 luminal domain
    • Complex: Homodimer of the luminal domain of PLD3
      • Protein or peptide: 5'-3' exonuclease PLD4
    • Complex: ssDNA
      • DNA: DNA (5'-D(*TP*TP*T)-3')
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: THYMIDINE-3'-PHOSPHATE
KeywordsExonuclease / IMMUNE SYSTEM
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
5'-3' exonuclease PLD4
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 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_64925.png

Downloads & links

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Map

FileDownload / File: emd_64925.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
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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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.073515974 - 0.1719883
Average (Standard dev.)0.00050825014 (±0.0056908936)
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_64925_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64925_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 : Homo-tetramer of PLD4 luminal domain

EntireName: Homo-tetramer of PLD4 luminal domain
Components
  • Complex: Homo-tetramer of PLD4 luminal domain
    • Complex: Homodimer of the luminal domain of PLD3
      • Protein or peptide: 5'-3' exonuclease PLD4
    • Complex: ssDNA
      • DNA: DNA (5'-D(*TP*TP*T)-3')
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: THYMIDINE-3'-PHOSPHATE

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Supramolecule #1: Homo-tetramer of PLD4 luminal domain

SupramoleculeName: Homo-tetramer of PLD4 luminal domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Homodimer of the luminal domain of PLD3

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

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others) / Synthetically produced: Yes

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Macromolecule #1: 5'-3' exonuclease PLD4

MacromoleculeName: 5'-3' exonuclease PLD4 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: spleen exonuclease
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.287273 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: RDSCQLVLVE SIPQDLPSAA GSPSAQPLGQ AWLQLLDTAQ ESVHVASYYW SLTGPDIGVN DSSSQLGEAL LQKLQQLLGR NISLAVATS SPTLARTSTD LQVLAARGAH VRQVPMGRLT RGVLHSKFWV VDGRHIYMGS ANMDWRSLTQ VKELGAVIYN C SHLAQDLE ...String:
RDSCQLVLVE SIPQDLPSAA GSPSAQPLGQ AWLQLLDTAQ ESVHVASYYW SLTGPDIGVN DSSSQLGEAL LQKLQQLLGR NISLAVATS SPTLARTSTD LQVLAARGAH VRQVPMGRLT RGVLHSKFWV VDGRHIYMGS ANMDWRSLTQ VKELGAVIYN C SHLAQDLE KTFQTYWVLG VPKAVLPKTW PQNFSSHFNR FQPFHGLFDG VPTTAYFSAS PPALCPQGRT RDLEALLAVM GS AQEFIYA SVMEYFPTTR FSHPPRYWPV LDNALRAAAF GKGVRVRLLV GCGLNTDPTM FPYLRSLQAL SNPAANVSVD VKV FIVPVG NHSNIPFSRV NHSKFMVTEK AAYIGTSNWS EDYFSSTAGV GLVVTQSPGA QPAGATVQEQ LRQLFERDWS SRYA VGLDG QAPGQDCVWQ GLEVLFQ

UniProtKB: 5'-3' exonuclease PLD4

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Macromolecule #2: DNA (5'-D(*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.86049 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 15 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: THYMIDINE-3'-PHOSPHATE

MacromoleculeName: THYMIDINE-3'-PHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: T3P
Molecular weightTheoretical: 322.208 Da
Chemical component information

ChemComp-ATD:
THYMIDINE-3'-PHOSPHATE

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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
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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 65742
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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