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Yorodumi- PDB-7tu8: Structure of the L. blandensis dGTPase H125A mutant bound to dGTP... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7tu8 | ||||||||||||
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Title | Structure of the L. blandensis dGTPase H125A mutant bound to dGTP and dATP | ||||||||||||
Components | dGTP triphosphohydrolase | ||||||||||||
Keywords | HYDROLASE / dGTPase / nucleotide binding | ||||||||||||
Function / homology | Function and homology information | ||||||||||||
Biological species | Leeuwenhoekiella blandensis MED217 (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å | ||||||||||||
Authors | Klemm, B.P. / Sikkema, A.P. / Hsu, A.L. / Borgnia, M.J. / Schaaper, R.M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: J Biol Chem / Year: 2022 Title: High-resolution structures of the SAMHD1 dGTPase homolog from Leeuwenhoekiella blandensis reveal a novel mechanism of allosteric activation by dATP. Authors: Bradley P Klemm / Andrew P Sikkema / Allen L Hsu / James C Horng / Traci M Tanaka Hall / Mario J Borgnia / Roel M Schaaper / Abstract: Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to ...Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7tu8.cif.gz | 601.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7tu8.ent.gz | 469.2 KB | Display | PDB format |
PDBx/mmJSON format | 7tu8.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7tu8_validation.pdf.gz | 1.9 MB | Display | wwPDB validaton report |
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Full document | 7tu8_full_validation.pdf.gz | 1.9 MB | Display | |
Data in XML | 7tu8_validation.xml.gz | 74.1 KB | Display | |
Data in CIF | 7tu8_validation.cif.gz | 112.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/tu/7tu8 ftp://data.pdbj.org/pub/pdb/validation_reports/tu/7tu8 | HTTPS FTP |
-Related structure data
Related structure data | 26129MC 7tu0C 7tu1C 7tu2C 7tu3C 7tu4C 7tu5C 7tu6C 7tu7C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 52656.176 Da / Num. of mol.: 6 / Mutation: H125A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Leeuwenhoekiella blandensis MED217 (bacteria) Strain: CECT 7118 / CCUG 51940 / MED217 / Gene: MED217_16760 / Plasmid: pMCSG7 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A3XHN1, dGTPase #2: Chemical | ChemComp-DGT / #3: Chemical | ChemComp-DTP / #4: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: dGTP triphosphohydrolase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Leeuwenhoekiella blandensis MED217 (bacteria) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pMCSG7 | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Hexamer concentration is listed. 10 mM dGTP and 10 mM dATP were added to the peak fraction after gel filtration. | ||||||||||||||||||||
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: 295 K / Details: 2.5 second blot time (front) |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
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: 279 |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 60 |
-Processing
EM software |
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Image processing | Details: 256 micrographs used after manually eliminating micrographs with poor CTF fit. | ||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 180962 / Details: Laplacian-of-Gaussian auto-picking | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D3 (2x3 fold dihedral) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94851 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL Details: Apo Cryo-EM model was fit into the EM map for building. dGTP/dATP ligands were initially built into the density using Coot. |