[English] 日本語
Yorodumi- EMDB-26362: Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26362 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Structure of E. coli dGTPase bound to T7 bacteriophage protein Gp1.2 and GTP | ||||||||||||
Map data | DeepEMhancer post-processed map | ||||||||||||
Sample |
| ||||||||||||
Keywords | dGTPase / inhibitor / complex / HYDROLASE | ||||||||||||
Function / homology | Function and homology information 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 | Escherichia coli str. K-12 substr. MG1655 (bacteria) / Escherichia phage T7 (virus) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.5 Å | ||||||||||||
Authors | Klemm BP / Hsu AL / Borgnia MJ / Schaaper RM | ||||||||||||
Funding support | United States, 3 items
| ||||||||||||
Citation | Journal: 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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_26362.map.gz | 40.9 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-26362-v30.xml emd-26362.xml | 29.3 KB 29.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26362_fsc.xml | 8.3 KB | Display | FSC data file |
Images | emd_26362.png | 136.8 KB | ||
Masks | emd_26362_msk_1.map | 47.6 MB | Mask map | |
Filedesc metadata | emd-26362.cif.gz | 7.3 KB | ||
Others | emd_26362_additional_1.map.gz emd_26362_additional_2.map.gz emd_26362_additional_3.map.gz emd_26362_half_map_1.map.gz emd_26362_half_map_2.map.gz | 36.5 MB 7.7 MB 44 MB 36.8 MB 36.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26362 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26362 | HTTPS FTP |
-Validation report
Summary document | emd_26362_validation.pdf.gz | 843.8 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_26362_full_validation.pdf.gz | 843.3 KB | Display | |
Data in XML | emd_26362_validation.xml.gz | 14 KB | Display | |
Data in CIF | emd_26362_validation.cif.gz | 20 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26362 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26362 | HTTPS FTP |
-Related structure data
Related structure data | 7u67MC 7u65C 7u66C M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_26362.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | DeepEMhancer post-processed map | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.932 Å | ||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | emd_26362_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: Full map from RELION refinement
File | emd_26362_additional_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Full map from RELION refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: RELION post-processed map
File | emd_26362_additional_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | RELION post-processed map | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: PHENIX auto-sharpened map
File | emd_26362_additional_3.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | PHENIX auto-sharpened map | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half-map 1
File | emd_26362_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half-map 1 | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half-map 2
File | emd_26362_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half-map 2 | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : dGTPase hexamer bound to six copies of Gp1.2
Entire | Name: dGTPase hexamer bound to six copies of Gp1.2 |
---|---|
Components |
|
-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: #1-#2 |
---|---|
Source (natural) | Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria) |
-Supramolecule #2: dGTP triphosphohydrolase
Supramolecule | Name: dGTP triphosphohydrolase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
---|---|
Source (natural) | Organism: Escherichia coli str. K-12 substr. MG1655 (bacteria) |
-Supramolecule #3: Gene 1.2 protein
Supramolecule | Name: Gene 1.2 protein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
---|---|
Source (natural) | Organism: Escherichia phage T7 (virus) |
-Macromolecule #1: Deoxyguanosinetriphosphate triphosphohydrolase
Macromolecule | Name: 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 weight | Theoretical: 59.470863 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
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 |
---|---|
Source (natural) | Organism: Escherichia phage T7 (virus) |
Molecular weight | Theoretical: 10.595868 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: GSFTMGRLYS GNLAAFKAAT NKLFQLDLAV IYDDWYDAYT RKDCIRLRIE DRSGNLIDTS TFYHHDEDVL FNMCTDWLNH MYDQLKDWK UniProtKB: Inhibitor of dGTPase |
-Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 6 / Formula: GTP |
---|---|
Molecular weight | Theoretical: 523.18 Da |
Chemical component information | ChemComp-GTP: |
-Macromolecule #4: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG |
---|---|
Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 Component:
| |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
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) along with 1 mM GTP |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
---|---|
Details | Defocus values as determined by CTFFIND-4.1 |
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: 1108 / Average exposure time: 8.4 sec. / Average electron dose: 54.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
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 |
+Image processing
-Atomic model buiding 1
Details | The dGTPase-Gp1.2 cryo-EM model was fit into the EM map, then the GTP built in using Coot. |
---|---|
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-7u67: |