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- PDB-7u65: Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 -

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Basic information

Entry
Database: PDB / ID: 7u65
TitleStructure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2
Components
  • Deoxyguanosinetriphosphate triphosphohydrolase
  • Inhibitor of dGTPase
KeywordsHYDROLASE / dGTPase / inhibitor / complex
Function / homology
Function and homology information


dGTPase / dGTPase activity / dGTP catabolic process / nucleobase-containing small molecule interconversion / cobalt ion binding / single-stranded DNA binding / manganese ion binding / DNA replication / GTPase activity / magnesium ion binding / identical protein binding
Similarity search - Function
Inhibitor of dGTPase, bacteriophage T7-like / Bacteriophage T7-like, gene 1.2 / dNTP triphosphohydrolase, type 1 / Deoxyguanosinetriphosphate triphosphohydrolase, central domain superfamily / dNTP triphosphohydrolase / HD domain profile. / HD domain / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
Inhibitor of dGTPase / Deoxyguanosinetriphosphate triphosphohydrolase
Similarity search - Component
Biological speciesEscherichia coli str. K-12 substr. MG1655 (bacteria)
Escherichia phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsKlemm, B.P. / Hsu, A.L. / Borgnia, M.J. / Schaaper, R.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES102906 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZICES103326 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIAES101905 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Mechanism by which T7 bacteriophage protein Gp1.2 inhibits dGTPase.
Authors: Bradley P Klemm / Deepa Singh / Cassandra E Smith / Allen L Hsu / Lucas B Dillard / Juno M Krahn / Robert E London / Geoffrey A Mueller / Mario J Borgnia / Roel M Schaaper /
Abstract: Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. encodes a dGTPase (2'- ...Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.
History
DepositionMar 3, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 31, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Deoxyguanosinetriphosphate triphosphohydrolase
G: Inhibitor of dGTPase
B: Deoxyguanosinetriphosphate triphosphohydrolase
C: Deoxyguanosinetriphosphate triphosphohydrolase
D: Deoxyguanosinetriphosphate triphosphohydrolase
E: Deoxyguanosinetriphosphate triphosphohydrolase
F: Deoxyguanosinetriphosphate triphosphohydrolase
H: Inhibitor of dGTPase
I: Inhibitor of dGTPase
J: Inhibitor of dGTPase
K: Inhibitor of dGTPase
L: Inhibitor of dGTPase


Theoretical massNumber of molelcules
Total (without water)420,40012
Polymers420,40012
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, The observed particles appear to be approximately hexameric and a separate C1 refinement confirmed that six copies of Gp1.2 are bound to the dGTPase hexamer.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Deoxyguanosinetriphosphate triphosphohydrolase / dGTP triphosphohydrolase / dGTPase


Mass: 59470.863 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli str. K-12 substr. MG1655 (bacteria)
Strain: K12 / Gene: dgt, b0160, JW0156 / Plasmid: pET30-Ek / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15723, dGTPase
#2: Protein
Inhibitor of dGTPase / Gene product 1.2 / Gp1.2 / Inhibitor of dGTPase gp1.2


Mass: 10595.868 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: The additional N-terminal sequence is retained after cleavage of the expression tag with TEV protease.
Source: (gene. exp.) Escherichia phage T7 (virus) / Gene: 1.2 / Plasmid: pDest-566 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03780

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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

Component
IDNameTypeEntity IDParent-IDSource
1dGTPase hexamer bound to six copies of Gp1.2COMPLEXall0MULTIPLE SOURCES
2dGTP triphosphohydrolaseDGTPaseCOMPLEX#11RECOMBINANT
3Gene 1.2 proteinCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli str. K-12 substr. MG1655 (bacteria)511145
22Escherichia coli str. K-12 substr. MG1655 (bacteria)511145
33Escherichia phage T7 (virus)10760
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
21Escherichia coli BL21(DE3) (bacteria)469008Multiple
22Escherichia coli BL21(DE3) (bacteria)469008pET30-Ek
33Escherichia coli BL21(DE3) (bacteria)469008pDest-566
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTristris(hydroxymethyl)aminomethane1
275 mMSodium citrateNa3C6H5O71
310 mMMagnesium chlorideMgCl21
41 mMbeta-mercaptoethanol2-Mercaptoethanol2-mercaptoethanol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Frozen stocks were thawed and mixed to a final concentration of 1.25:1 Gp1.2 to dGTPase (monomer)
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 290 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 1750 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8.4 sec. / Electron dose: 54 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1638
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
4CTFFIND4.1CTF correction
7PHENIXmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
Image processingDetails: 1404 micrographs used after manually eliminating micrographs with poor CTF fit.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 732832 / Details: Laplacian-of-Gaussian auto-picking
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114127 / Algorithm: FOURIER SPACE / Num. of class averages: 93 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: E. coli dGTPase crystal structure (PDB ID: 4XDS) and T7 bacteriophage NMR structure (PDB ID: 2MDP) models were fit into the EM map using Chimera for subsequent building. After an initial ...Details: E. coli dGTPase crystal structure (PDB ID: 4XDS) and T7 bacteriophage NMR structure (PDB ID: 2MDP) models were fit into the EM map using Chimera for subsequent building. After an initial round of real-space refinement, the Gp1.2 N-terminus was re-built into the density using Coot.
Atomic model building
IDPDB-ID 3D fitting-ID
14XDS

4xds

1
22MDP

2mdp

1

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