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- EMDB-26361: Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp... -

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Basic information

Entry
Database: EMDB / ID: EMD-26361
TitleStructure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 and dGTP
Map dataDeepEMhancer post-processed map
Sample
  • Complex: dGTPase hexamer bound to six copies of Gp1.2
    • Complex: dGTP triphosphohydrolaseDGTPase
      • Protein or peptide: Deoxyguanosinetriphosphate triphosphohydrolase
    • Complex: Gene 1.2 protein
      • Protein or peptide: Inhibitor of dGTPase
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsKlemm BP / Dillard LB / Borgnia MJ / Schaaper RM
Funding support United States, 3 items
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, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26361.png

Downloads & links

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Map

FileDownload / File: emd_26361.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer post-processed map
Voxel sizeX=Y=Z: 0.932 Å
Density
Contour LevelBy AUTHOR: 0.199
Minimum - Maximum-0.03305063 - 1.8492329
Average (Standard dev.)0.005818418 (±0.054332744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 216.224 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26361_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION post-processed map

Fileemd_26361_additional_1.map
AnnotationRELION post-processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: PHENIX auto-sharpened map

Fileemd_26361_additional_2.map
AnnotationPHENIX auto-sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Full map from RELION refinement

Fileemd_26361_additional_3.map
AnnotationFull map from RELION refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_26361_half_map_1.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_26361_half_map_2.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dGTPase hexamer bound to six copies of Gp1.2

EntireName: dGTPase hexamer bound to six copies of Gp1.2
Components
  • Complex: dGTPase hexamer bound to six copies of Gp1.2
    • Complex: dGTP triphosphohydrolaseDGTPase
      • Protein or peptide: Deoxyguanosinetriphosphate triphosphohydrolase
    • Complex: Gene 1.2 protein
      • Protein or peptide: Inhibitor of dGTPase
  • Ligand: MAGNESIUM ION
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: dGTPase hexamer bound to six copies of Gp1.2

SupramoleculeName: dGTPase hexamer bound to six copies of Gp1.2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)

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Supramolecule #2: dGTP triphosphohydrolase

SupramoleculeName: dGTP triphosphohydrolase / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET30-Ek

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Supramolecule #3: Gene 1.2 protein

SupramoleculeName: Gene 1.2 protein / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia phage T7 (virus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pDest-566

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Macromolecule #1: Deoxyguanosinetriphosphate triphosphohydrolase

MacromoleculeName: Deoxyguanosinetriphosphate triphosphohydrolase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: dGTPase
Source (natural)Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria)
Strain: K12
Molecular weightTheoretical: 59.470863 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAQIDFRKKI NWHRRYRSPQ GVKTEHEILR IFESDRGRII NSPAIRRLQQ KTQVFPLERN AAVRTRLTHS MEVQQVGRYI AKEILSRLK ELKLLEAYGL DELTGPFESI VEMSCLMHDI GNPPFGHFGE AAINDWFRQR LHPEDAESQP LTDDRCSVAA L RLRDGEEP ...String:
MAQIDFRKKI NWHRRYRSPQ GVKTEHEILR IFESDRGRII NSPAIRRLQQ KTQVFPLERN AAVRTRLTHS MEVQQVGRYI AKEILSRLK ELKLLEAYGL DELTGPFESI VEMSCLMHDI GNPPFGHFGE AAINDWFRQR LHPEDAESQP LTDDRCSVAA L RLRDGEEP LNELRRKIRQ DLCHFEGNAQ GIRLVHTLMR MNLTWAQVGG ILKYTRPAWW RGETPETHHY LMKKPGYYLS EE AYIARLR KELNLALYSR FPLTWIMEAA DDISYCVADL EDAVEKRIFT VEQLYHHLHE AWGQHEKGSL FSLVVENAWE KSR SNSLSR STEDQFFMYL RVNTLNKLVP YAAQRFIDNL PAIFAGTFNH ALLEDASECS DLLKLYKNVA VKHVFSHPDV ERLE LQGYR VISGLLEIYR PLLSLSLSDF TELVEKERVK RFPIESRLFH KLSTRHRLAY VEAVSKLPSD SPEFPLWEYY YRCRL LQDY ISGMTDLYAW DEYRRLMAVE Q

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Macromolecule #2: Inhibitor of dGTPase

MacromoleculeName: Inhibitor of dGTPase / type: protein_or_peptide / ID: 2
Details: The additional N-terminal sequence is retained after cleavage of the expression tag with TEV protease.
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage T7 (virus)
Molecular weightTheoretical: 10.595868 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSFTMGRLYS GNLAAFKAAT NKLFQLDLAV IYDDWYDAYT RKDCIRLRIE DRSGNLIDTS TFYHHDEDVL FNMCTDWLNH MYDQLKDWK

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: DGT
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-DGT:
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / Deoxyguanosine triphosphate

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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: 8
Component:
ConcentrationFormulaName
25.0 mMtris(hydroxymethyl)aminomethaneTris
75.0 mMNa3C6H5O7Sodium citrate
10.0 mMMgCl2Magnesium chloride
1.0 mM2-mercaptoethanolbeta-mercaptoethanol2-Mercaptoethanol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 290 K / Instrument: LEICA EM GP
DetailsFrozen stocks were thawed and mixed to a final concentration of 1.25:1 Gp1.2 to dGTPase (monomer) along with 1 mM dGTP

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 1206 / Average exposure time: 8.4 sec. / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 496081 / Details: Laplacian-of-Gaussian auto-picking
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: INSILICO MODEL
In silico model: Initial model from the dGTPase-Gp1.2-GTP dataset
Details: Low-pass filtered to ~60 angstroms
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 200 / Avg.num./class: 617 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 100 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 113934
Details1037 micrographs used after eliminating micrographs with a CTF fit > 4.5 angstroms.
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe dGTPase-Gp1.2 cryo-EM model was fit into the EM map, then the dGTP built in using Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7u66:
Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 and dGTP

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