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- PDB-9p8t: Structure of CloA co-expressed with CloB -

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Basic information

Entry
Database: PDB / ID: 9p8t
TitleStructure of CloA co-expressed with CloB
Components
  • 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
  • DNTP 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
DNA / DNTP triphosphohydrolase
Similarity search - Component
Biological speciesSalmonella enterica (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.65 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
Revision 1.0Jul 30, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 30, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 2.0Feb 11, 2026Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Refinement description / Structure summary
Category: atom_site / database_PDB_caveat ...atom_site / database_PDB_caveat / em_admin / em_entity_assembly / em_software / pdbx_contact_author / pdbx_poly_seq_scheme / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_residues / pdbx_validate_chiral / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / refine / refine_ls_restr / refine_ls_restr_ncs / struct_conf / struct_conn / struct_ncs_dom / struct_ncs_dom_lim / struct_ncs_oper
Item: _em_admin.last_update / _em_entity_assembly.entity_id_list ..._em_admin.last_update / _em_entity_assembly.entity_id_list / _em_software.fitting_id / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _pdbx_validate_rmsd_angle.angle_deviation / _pdbx_validate_rmsd_angle.angle_value / _refine.B_iso_mean / _refine_ls_restr.dev_ideal / _refine_ls_restr.number / _refine_ls_restr_ncs.pdbx_asym_id / _refine_ls_restr_ncs.pdbx_auth_asym_id / _refine_ls_restr_ncs.rms_dev_position / _struct_conf.beg_auth_comp_id / _struct_conf.beg_auth_seq_id / _struct_conf.beg_label_comp_id / _struct_conf.beg_label_seq_id / _struct_conf.end_auth_comp_id / _struct_conf.end_auth_seq_id / _struct_conf.end_label_comp_id / _struct_conf.end_label_seq_id / _struct_conf.pdbx_PDB_helix_length / _struct_ncs_dom.details / _struct_ncs_oper.matrix[1][1] / _struct_ncs_oper.matrix[1][2] / _struct_ncs_oper.matrix[1][3] / _struct_ncs_oper.matrix[2][1] / _struct_ncs_oper.matrix[2][2] / _struct_ncs_oper.matrix[2][3] / _struct_ncs_oper.matrix[3][1] / _struct_ncs_oper.matrix[3][2] / _struct_ncs_oper.matrix[3][3] / _struct_ncs_oper.vector[1] / _struct_ncs_oper.vector[2] / _struct_ncs_oper.vector[3]
Description: Chirality error / Provider: author / Type: Coordinate replacement
Revision 1.1Feb 11, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / EM metadata
Group: Data processing / Experimental summary / Source and taxonomy
Data content type: EM metadata / EM metadata / EM metadata / Category: em_admin / em_entity_assembly / em_software
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_entity_assembly.entity_id_list ..._em_admin.last_update / _em_entity_assembly.entity_id_list / _em_software.fitting_id / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name

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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
M: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
N: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
O: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
P: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
Q: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
R: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
S: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
T: 5'-diphosphothymidyl-3'5'-thymidine (p2diT)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)440,40024
Polymers440,20516
Non-polymers1948
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "G"
d_2ens_1chain "B"
d_3ens_1chain "D"
d_4ens_1chain "E"
d_5ens_1chain "J"
d_6ens_1chain "H"
d_7ens_1chain "A"
d_8ens_1chain "K"

NCS domain segments:

Component-ID: 1 / Ens-ID: ens_1 / Beg auth comp-ID: SER / Beg label comp-ID: SER / End auth comp-ID: CYS / End label comp-ID: CYS / Auth seq-ID: 0 - 468 / Label seq-ID: 2 - 470

Dom-IDAuth asym-IDLabel asym-ID
d_1GE
d_2BB
d_3DC
d_4ED
d_5JG
d_6HF
d_7AA
d_8KH

NCS oper:
IDCodeMatrixVector
1given(-0.913456335442, 0.406936737829, -0.000121020191851), (0.406936566432, 0.913456156838, 0.000693134922817), (0.000392608703715, 0.000583900945222, -0.999999752459)199.857311143, -42.5841774577, 254.215979118
2given(-2.22044604925E-16, 1), (-1, -2.22044604925E-16), (1)-1.42108547152E-13, 265.32, 6.25277607469E-13
3given(-0.406936566432, -0.913456156838, -0.000693134922816), (-0.913456335442, 0.406936737829, -0.000121020191853), (0.000392608703716, 0.000583900945221, -0.999999752459)307.904177458, 199.857311143, 254.215979118
4given(-1.02937286195E-6, -0.999999999992, -3.77535994296E-6), (0.999999999999, -1.02937267132E-6, -5.04744235727E-8), (5.047053732E-8, -3.77535999491E-6, 0.999999999993)265.320210031, 0.00014940216461, 0.000516936479983
5given(0.913456335442, -0.406936737829, 0.000121020191852), (-0.406936566432, -0.913456156838, -0.000693134922816), (0.000392608703715, 0.000583900945221, -0.999999752459)65.4626888567, 307.904177458, 254.215979118
6given(-1), (-1), (1)265.32, 265.32, 3.69482222595E-13
7given(0.406937544547, 0.913455718969, 0.000695932295615), (0.91345589917, -0.406937717103, 0.000121120122386), (0.000393838968111, 0.000586415135654, -0.999999750504)-42.5850574423, 65.4628504464, 254.215517253

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Components

#1: Protein
DNTP triphosphohydrolase


Mass: 54382.273 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: DNA chain
5'-diphosphothymidyl-3'5'-thymidine (p2diT)


Mass: 643.408 Da / Num. of mol.: 8 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY
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: MULTIPLE SOURCES
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: 600 nm
Image recordingElectron dose: 48.69 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
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 221450 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 114.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001631240
ELECTRON MICROSCOPYf_angle_d0.395742176
ELECTRON MICROSCOPYf_chiral_restr0.03324504
ELECTRON MICROSCOPYf_plane_restr0.00265456
ELECTRON MICROSCOPYf_dihedral_angle_d6.81824240
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2EGELECTRON MICROSCOPYNCS constraints6.83237394988E-13
ens_1d_3EGELECTRON MICROSCOPYNCS constraints2.03371667276E-11
ens_1d_4EGELECTRON MICROSCOPYNCS constraints4.45957048529E-13
ens_1d_5EGELECTRON MICROSCOPYNCS constraints1.99727869872E-11
ens_1d_6EGELECTRON MICROSCOPYNCS constraints3.55695286884E-13
ens_1d_7EGELECTRON MICROSCOPYNCS constraints3.41445487178E-11
ens_1d_8EGELECTRON MICROSCOPYNCS constraints6.89132599972E-13

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