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- EMDB-26360: Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 -
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Open data
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Basic information
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Title | Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 | ||||||||||||
![]() | DeepEMhancer post-processed map | ||||||||||||
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![]() | dGTPase / inhibitor / complex / HYDROLASE | ||||||||||||
Function / homology | ![]() 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 | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | ||||||||||||
![]() | Klemm BP / Hsu AL / Borgnia MJ / Schaaper RM | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 41.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 29.1 KB 29.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 8.3 KB | Display | ![]() |
Images | ![]() | 148.8 KB | ||
Masks | ![]() | 47.6 MB | ![]() | |
Filedesc metadata | ![]() | 7.2 KB | ||
Others | ![]() ![]() ![]() ![]() ![]() | 8.6 MB 44 MB 36.5 MB 36.7 MB 36.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 850.1 KB | Display | ![]() |
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Full document | ![]() | 849.7 KB | Display | |
Data in XML | ![]() | 14 KB | Display | |
Data in CIF | ![]() | 19.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7u65MC ![]() 7u66C ![]() 7u67C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | DeepEMhancer post-processed map | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.932 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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-Additional map: RELION post-processed map
File | emd_26360_additional_1.map | ||||||||||||
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Annotation | RELION post-processed map | ||||||||||||
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-Additional map: PHENIX auto-sharpened map
File | emd_26360_additional_2.map | ||||||||||||
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Annotation | PHENIX auto-sharpened map | ||||||||||||
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-Additional map: Full map from RELION refinement
File | emd_26360_additional_3.map | ||||||||||||
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Annotation | Full map from RELION refinement | ||||||||||||
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Density Histograms |
-Half map: Half-map 1
File | emd_26360_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 | ||||||||||||
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-Half map: Half-map 2
File | emd_26360_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 | ||||||||||||
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Sample components
-Entire : dGTPase hexamer bound to six copies of Gp1.2
Entire | Name: dGTPase hexamer bound to six copies of Gp1.2 |
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Components |
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-Supramolecule #1: dGTPase hexamer bound to six copies of Gp1.2
Supramolecule | Name: dGTPase hexamer bound to six copies of Gp1.2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #2: dGTP triphosphohydrolase
Supramolecule | Name: dGTP triphosphohydrolase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: Gene 1.2 protein
Supramolecule | Name: Gene 1.2 protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Deoxyguanosinetriphosphate triphosphohydrolase
Macromolecule | Name: Deoxyguanosinetriphosphate triphosphohydrolase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: dGTPase |
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Source (natural) | Organism: ![]() ![]() Strain: K12 |
Molecular weight | Theoretical: 59.470863 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 UniProtKB: Deoxyguanosinetriphosphate triphosphohydrolase |
-Macromolecule #2: Inhibitor of dGTPase
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 10.595868 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: GSFTMGRLYS GNLAAFKAAT NKLFQLDLAV IYDDWYDAYT RKDCIRLRIE DRSGNLIDTS TFYHHDEDVL FNMCTDWLNH MYDQLKDWK UniProtKB: Inhibitor of dGTPase |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 8 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 290 K / Instrument: LEICA EM GP | |||||||||||||||
Details | Frozen stocks were thawed and mixed to a final concentration of 1.25:1 Gp1.2 to dGTPase (monomer) |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film 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: 1638 / Average exposure time: 8.4 sec. / Average electron dose: 54.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.75 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 45000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model |
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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. | ||||||
Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||
Output model | ![]() PDB-7u65: |