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- PDB-9p8s: Structure of CloA apo -

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Basic information

Entry
Database: PDB / ID: 9p8s
TitleStructure of CloA apo
ComponentsDNTP triphosphohydrolase
KeywordsHYDROLASE / deoxynucleoside triphosphohydrolase
Function / homology
Function and homology information


dGTPase activity / dGTP catabolic process
Similarity search - Function
Deoxyguanosinetriphosphate triphosphohydrolase, C-terminal / Deoxyguanosinetriphosphate triphosphohydrolase, central domain superfamily / dNTP triphosphohydrolase / : / HD domain / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
DNTP triphosphohydrolase
Similarity search - Component
Biological speciesSalmonella enterica (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsYamaguchi, S. / Fernandez, S.G. / Wassarman, D.R. / Luder, M. / Schwede, F. / Kranzusch, P.J.
Funding support Japan, France, United States, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)202360072 Japan
Human Frontier Science Program (HFSP)LT0051 France
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2 GM146250-01 United States
CitationJournal: bioRxiv / Year: 2025
Title: Activating and inhibiting nucleotide signals coordinate bacterial anti-phage defense.
Authors: Sonomi Yamaguchi / Samantha G Fernandez / Douglas R Wassarman / Marlen Lüders / Frank Schwede / Philip J Kranzusch
Abstract: The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool allow animal and bacterial cells to ...The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool allow animal and bacterial cells to broadly restrict diverse viruses, but reduced nucleotide availability induces cellular toxicity and can limit host fitness(Ahmad et al., 1998; Goldstone et al., 2011; Hsueh et al., 2022; Itsko & Schaaper, 2014; Tal et al., 2022). Here we discover a bacterial anti-phage defense system named Clover that overcomes this tradeoff by encoding a deoxynucleoside triphosphohydrolase enzyme (CloA) that dynamically responds to both an activating phage cue and an inhibitory nucleotide immune signal produced by a partnering regulatory enzyme (CloB). Analysis of Clover phage restriction in cells and reconstitution of enzymatic function in vitro demonstrate that CloA is a dGTPase that responds to viral enzymes that increase cellular levels of dTTP. To restrain CloA activation in the absence of infection, we show that CloB synthesizes a dTTP-related inhibitory nucleotide signal p3diT (5'-triphosphothymidyl-3'5'-thymidine) that binds to CloA and suppresses activation. Cryo-EM structures of CloA in activated and suppressed states reveal how dTTP and p3diT control distinct allosteric sites and regulate effector function. Our results define how nucleotide signals coordinate both activation and inhibition of antiviral immunity and explain how cells balance defense and immune-mediated toxicity.
History
DepositionJun 23, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNTP triphosphohydrolase
B: DNTP triphosphohydrolase
D: DNTP triphosphohydrolase
E: DNTP triphosphohydrolase
G: DNTP triphosphohydrolase
H: DNTP triphosphohydrolase
J: DNTP triphosphohydrolase
K: DNTP triphosphohydrolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)434,90016
Polymers434,7068
Non-polymers1948
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
DNTP triphosphohydrolase


Mass: 54338.223 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica (bacteria) / Gene: dgt, ECD07_17535, EIW74_15545, GB147_17355 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A5H6DAK1
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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 Clover A / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Salmonella enterica (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 49.66 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21_5207model refinement
13cryoSPARC4.5.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324832 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 103.81 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002629836
ELECTRON MICROSCOPYf_angle_d0.427340192
ELECTRON MICROSCOPYf_chiral_restr0.03334292
ELECTRON MICROSCOPYf_plane_restr0.00325248
ELECTRON MICROSCOPYf_dihedral_angle_d3.77093976

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