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- PDB-9p8w: Anti-phage dGTPase from Shewanella putrefaciens CN-32 -

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Basic information

Entry
Database: PDB / ID: 9p8w
TitleAnti-phage dGTPase from Shewanella putrefaciens CN-32
ComponentsDeoxyguanosinetriphosphate triphosphohydrolase-like protein
KeywordsHYDROLASE / deoxynucleoside triphosphohydrolase
Function / homology
Function and homology information


dGTPase activity / dGTP catabolic process
Similarity search - Function
dNTP triphosphohydrolase, type 2 / Phosphohydrolase-associated domain / Phosphohydrolase-associated domain / dNTP triphosphohydrolase / : / HD domain profile. / HD domain / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
Deoxyguanosinetriphosphate triphosphohydrolase-like protein
Similarity search - Component
Biological speciesShewanella putrefaciens CN-32 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.68 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

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Assembly

Deposited unit
A: Deoxyguanosinetriphosphate triphosphohydrolase-like protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,4252
Polymers53,4011
Non-polymers241
Water1,820101
1
A: Deoxyguanosinetriphosphate triphosphohydrolase-like protein
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)320,55012
Polymers320,4046
Non-polymers1466
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_545-y,x-y-1,z1
crystal symmetry operation3_655-x+y+1,-x,z1
crystal symmetry operation10_554-y,-x,-z-1/21
crystal symmetry operation11_654-x+y+1,y,-z-1/21
crystal symmetry operation12_544x,x-y-1,-z-1/21
Buried area25660 Å2
ΔGint-111 kcal/mol
Surface area105950 Å2
MethodPISA
Unit cell
Length a, b, c (Å)121.718, 121.718, 151.483
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Space group name HallP6c2c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: x-y,-y,-z
#7: -x,-x+y,-z
#8: -x,-y,z+1/2
#9: y,x,-z
#10: -y,-x,-z+1/2
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-620-

HOH

21A-701-

HOH

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Components

#1: Protein Deoxyguanosinetriphosphate triphosphohydrolase-like protein


Mass: 53400.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shewanella putrefaciens CN-32 (bacteria)
Gene: Sputcn32_0159 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A4Y1R2
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 101 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.03 Å3/Da / Density % sol: 59.45 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop
Details: 0.2 M magnesium chloride, 0.1 M HEPES-NaOH, pH 7.5, 30% PEG400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9792 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Feb 28, 2025
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.68→105.41 Å / Num. obs: 19233 / % possible obs: 99.9 % / Redundancy: 39 % / Biso Wilson estimate: 50.25 Å2 / CC1/2: 0.853 / Rmerge(I) obs: 0.378 / Rpim(I) all: 0.084 / Rrim(I) all: 0.387 / Rsym value: 0.378 / Net I/σ(I): 11.9
Reflection shellResolution: 2.68→2.81 Å / Redundancy: 40.1 % / Rmerge(I) obs: 2.97 / Mean I/σ(I) obs: 1.9 / Num. unique obs: 2502 / CC1/2: 0.853 / R split: 3.042 / Rpim(I) all: 0.658 / Rsym value: 2.97 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.21_5207refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.68→75.74 Å / SU ML: 0.3616 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.787
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2658 926 4.81 %
Rwork0.2084 18307 -
obs0.211 19233 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 60.54 Å2
Refinement stepCycle: LAST / Resolution: 2.68→75.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3676 0 1 101 3778
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00233753
X-RAY DIFFRACTIONf_angle_d0.48785052
X-RAY DIFFRACTIONf_chiral_restr0.0358540
X-RAY DIFFRACTIONf_plane_restr0.0033663
X-RAY DIFFRACTIONf_dihedral_angle_d12.01421441
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.68-2.820.341510.28252530X-RAY DIFFRACTION99.89
2.82-30.33661270.27232550X-RAY DIFFRACTION99.96
3-3.230.32091190.24932579X-RAY DIFFRACTION99.96
3.23-3.550.29281310.21592579X-RAY DIFFRACTION100
3.56-4.070.24751320.19472602X-RAY DIFFRACTION99.96
4.07-5.130.19371350.17272642X-RAY DIFFRACTION100
5.13-75.740.26541310.19362825X-RAY DIFFRACTION99.83
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.28903641826-0.2758421625050.2414003388831.487997563310.05201790234591.45931223117-0.146546501891-0.0511846568879-0.1011141916160.1310801592740.08855299344950.1324096567880.437353494897-0.3917121893380.03609224484540.633378050407-0.1214134108830.04424009249490.7414527527710.03460385403070.47432766755425.1769664357-57.463450872-46.3292099743
21.605350570560.230685329556-0.1189634126890.592849378699-0.01465869831061.25731271738-0.036280345193-0.09262064604870.08776293957140.08200671796520.02054899568610.0379244980196-0.0347127510615-0.2900190005880.003167339467740.395334670516-0.0219592430150.007004961899850.4313876244810.006731940647770.34323389213933.6122591014-43.7751819654-56.0132903341
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION / Auth asym-ID: A / Label asym-ID: A

IDRefine TLS-IDSelection detailsAuth seq-IDLabel seq-ID
11chain 'A' and (resid 1 through 98 )1 - 981 - 91
22chain 'A' and (resid 99 through 459 )99 - 45992 - 452

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