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Yorodumi- PDB-8v54: Engaged conformation of the human mitochondrial DNA polymerase ga... -
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-Basic information
Entry | Database: PDB / ID: 8v54 | ||||||||||||
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Title | Engaged conformation of the human mitochondrial DNA polymerase gamma bound to DNA | ||||||||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / Mitochondria DNA polymerase / DNA polymerase gamma / Mitochondrial DNA replication / DNA binding protein / DNA BINDING PROTEIN-DNA complex | ||||||||||||
Function / homology | Function and homology information gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / DNA replication proofreading / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases ...gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / DNA replication proofreading / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / DNA polymerase binding / base-excision repair, gap-filling / 3'-5' exonuclease activity / base-excision repair / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / protease binding / double-stranded DNA binding / in utero embryonic development / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mitochondrial matrix / intracellular membrane-bounded organelle / chromatin binding / protein-containing complex / mitochondrion / DNA binding / identical protein binding / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å | ||||||||||||
Authors | Riccio, A.A. / Krahn, J.M. / Bouvette, J. / Borgnia, J.M. / Copeland, W.C. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Nucleic Acids Res / Year: 2024 Title: Coordinated DNA polymerization by Polγ and the region of LonP1 regulated proteolysis. Authors: Amanda A Riccio / Asia J Brannon / Juno M Krahn / Jonathan Bouvette / Jason G Williams / Mario J Borgnia / William C Copeland / Abstract: The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions ...The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8v54.cif.gz | 408.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8v54.ent.gz | 319.1 KB | Display | PDB format |
PDBx/mmJSON format | 8v54.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8v54_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8v54_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8v54_validation.xml.gz | 64.4 KB | Display | |
Data in CIF | 8v54_validation.cif.gz | 96 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/v5/8v54 ftp://data.pdbj.org/pub/pdb/validation_reports/v5/8v54 | HTTPS FTP |
-Related structure data
Related structure data | 42979MC 8v55C 8v5dC 8v5rC 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: 138723.438 Da / Num. of mol.: 1 / Mutation: D198A, E200A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLG / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P54098 | ||||
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#2: Protein | Mass: 53787.281 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: POLG2, MTPOLB / Production host: Escherichia coli (E. coli) / Strain (production host): bl21DE3 / References: UniProt: Q9UHN1 #3: DNA chain | | Mass: 7595.886 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) #4: DNA chain | | Mass: 13597.686 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) |
-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: Complex of the human mitochondrial DNA Polymerase Gamma (POLG/POLG2 heterotrimer) bound to a DNA fork in an engaged conformation Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Value: 0.220 MDa / Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21 (De3) |
Buffer solution | pH: 8 |
Specimen | Conc.: 0.22 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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 defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
Image scans | Width: 3708 / Height: 3838 |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43451 / Symmetry type: POINT | ||||||||||||||||||||||||
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