[English] 日本語
![](img/lk-miru.gif)
- PDB-7u67: Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp... -
+
Open data
-
Basic information
Entry | Database: PDB / ID: 7u67 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 and GTP | ||||||||||||
![]() |
| ||||||||||||
![]() | HYDROLASE / dGTPase / inhibitor / complex | ||||||||||||
Function / homology | ![]() dGTPase / dGTPase activity / dGTP catabolic process / nucleobase-containing small molecule interconversion / cobalt ion binding / manganese ion binding / single-stranded DNA binding / DNA replication / GTPase activity / magnesium ion binding / identical protein binding Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å | ||||||||||||
![]() | Klemm, B.P. / Hsu, A.L. / Borgnia, M.J. / Schaaper, R.M. | ||||||||||||
Funding support | ![]()
| ||||||||||||
![]() | ![]() 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 |
|
-
Structure visualization
Structure viewer | Molecule: ![]() ![]() |
---|
-
Downloads & links
-
Download
PDBx/mmCIF format | ![]() | 760 KB | Display | ![]() |
---|---|---|---|---|
PDB format | ![]() | 583.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
---|
-Related structure data
Related structure data | ![]() 26362MC ![]() 7u65C ![]() 7u66C M: map data used to model this data C: citing same article ( |
---|---|
Similar structure data | Similarity search - Function & homology ![]() |
Other databases |
|
-
Links
-
Assembly
Deposited unit | ![]()
|
---|---|
1 |
|
-
Components
#1: Protein | Mass: 59470.863 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: K12 / Gene: dgt, b0160, JW0156 / Plasmid: pET30-Ek / Production host: ![]() ![]() #2: Protein | 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.) ![]() ![]() ![]() ![]() #3: Chemical | ChemComp-GTP / #4: Chemical | ChemComp-MG / Has ligand of interest | Y | |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-
Sample preparation
Component |
| |||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight |
| |||||||||||||||||||||||||
Source (natural) |
| |||||||||||||||||||||||||
Source (recombinant) |
| |||||||||||||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||||||||||||
Buffer component |
| |||||||||||||||||||||||||
Specimen | Embedding 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) along with 1 mM GTP | |||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 290 K |
-
Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TALOS ARCTICA / Details: Defocus values as determined by CTFFIND-4.1 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 1750 nm / Nominal defocus min: 750 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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: 1108 |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 60 |
-
Processing
EM software |
| ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Image processing | Details: 1004 micrographs used after manually eliminating micrographs with poor CTF fit. | ||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 568611 / Details: Laplacian-of-Gaussian auto-picking | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D3 (2x3 fold dihedral) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130940 / Algorithm: FOURIER SPACE / Num. of class averages: 119 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL Details: The dGTPase-Gp1.2 cryo-EM model was fit into the EM map, then the GTP built in using Coot. |