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- EMDB-27163: Human mitochondrial DNA polymerase gamma ternary complex with GT ... -

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Basic information

Entry
Database: EMDB / ID: EMD-27163
TitleHuman mitochondrial DNA polymerase gamma ternary complex with GT basepair in intermediate conformer
Map data
Sample
  • Complex: Human mitochondrial DNA polymerase gamma ternary complex with GT basepair
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*T)-3')
    • DNA: DNA (5'-D(P*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium
KeywordsDNA-binding protein / DNA polymerase / TRANSFERASE-DNA complex
Function / homology
Function and homology information


gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / 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 / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / 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 / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding / in utero embryonic development / protease binding / 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
DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. ...DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Anticodon-binding domain superfamily / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase subunit gamma-1 / DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsPark J / Yin YW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI134611 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.
Authors: Joon Park / Geoffrey K Herrmann / Patrick G Mitchell / Michael B Sherman / Y Whitney Yin /
Abstract: Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural ...Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.
History
DepositionJun 1, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27163.png

Downloads & links

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Map

FileDownload / File: emd_27163.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum0.0 - 10.976428
Average (Standard dev.)0.33908308 (±0.59695953)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: CryoSPARC unsharpened map

Fileemd_27163_additional_1.map
AnnotationCryoSPARC unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27163_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27163_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : Human mitochondrial DNA polymerase gamma ternary complex with GT ...

EntireName: Human mitochondrial DNA polymerase gamma ternary complex with GT basepair
Components
  • Complex: Human mitochondrial DNA polymerase gamma ternary complex with GT basepair
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*T)-3')
    • DNA: DNA (5'-D(P*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: Human mitochondrial DNA polymerase gamma ternary complex with GT ...

SupramoleculeName: Human mitochondrial DNA polymerase gamma ternary complex with GT basepair
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA polymerase subunit gamma-1

MacromoleculeName: DNA polymerase subunit gamma-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 139.730703 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA ...String:
MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA WAWAEGWTRY GPEGEAVPVA IPEERALVFD VEVCLAEGTC PTLAVAISPS AWYSWCSQRL VEERYSWTSQ LS PADLIPL EVPTGASSPT QRDWQEQLVV GHNVSFDRAH IREQYLIQGS RMRFLDTMSM HMAISGLSSF QRSLWIAAKQ GKH KVQPPT KQGQKSQRKA RRGPAISSWD WLDISSVNSL AEVHRLYVGG PPLEKEPREL FVKGTMKDIR ENFQDLMQYC AQDV WATHE VFQQQLPLFL ERCPHPVTLA GMLEMGVSYL PVNQNWERYL AEAQGTYEEL QREMKKSLMD LANDACQLLS GERYK EDPW LWDLEWDLQE FKQKKAKKVK KEPATASKLP IEGAGAPGDP MDQEDLGPCS EEEEFQQDVM ARACLQKLKG TTELLP KRP QHLPGHPGWY RKLCPRLDDP AWTPGPSLLS LQMRVTPKLM ALTWDGFPLH YSERHGWGYL VPGRRDNLAK LPTGTTL ES AGVVCPYRAI ESLYRKHCLE QGKQQLMPQE AGLAEEFLLT DNSAIWQTVE ELDYLEVEAE AKMENLRAAV PGQPLALT A RGGPKDTQPS YHHGNGPYND VDIPGCWFFK LPHKDGNSCN VGSPFAKDFL PKMEDGTLQA GPGGASGPRA LEINKMISF WRNAHKRISS QMVVWLPRSA LPRAVIRHPD YDEEGLYGAI LPQVVTAGTI TRRAVEPTWL TASNARPDRV GSELKAMVQA PPGYTLVGA DVDSQELWIA AVLGDAHFAG MHGCTAFGWM TLQGRKSRGT DLHSKTATTV GISREHAKIF NYGRIYGAGQ P FAERLLMQ FNHRLTQQEA AEKAQQMYAA TKGLRWYRLS DEGEWLVREL NLPVDRTEGG WISLQDLRKV QRETARKSQW KK WEVVAER AWKGGTESEM FNKLESIATS DIPRTPVLGC CISRALEPSA VQEEFMTSRV NWVVQSSAVD YLHLMLVAMK WLF EEFAID GRFCISIHDE VRYLVREEDR YRAALALQIT NLLTRCMFAY KLGLNDLPQS VAFFSAVDID RCLRKEVTMD CKTP SNPTG MERRYGIPQG EALDIYQIIE LTKGSLEKRS QPGP

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Macromolecule #2: DNA polymerase subunit gamma-2, mitochondrial

MacromoleculeName: DNA polymerase subunit gamma-2, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.991 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV ...String:
MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV AFLENVLKTS GKLRENLLHG ALEHYVNCLD LVNKRLPYGL AQIGVCFHPV FDTKQIRNGV KSIGEKTEAS LV WFTPPRT SNQWLDFWLR HRLQWWRKFA MSPSNFSSSD CQDEEGRKGN KLYYNFPWGK ELIETLWNLG DHELLHMYPG NVS KLHGRD GRKNVVPCVL SVNGDLDRGM LAYLYDSFQL TENSFTRKKN LHRKVLKLHP CLAPIKVALD VGRGPTLELR QVCQ GLFNE LLENGISVWP GYLETMQSSL EQLYSKYDEM SILFTVLVTE TTLENGLIHL RSRDTTMKEM MHISKLKDFL IKYIS SAKN V

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Macromolecule #3: DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP...

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.372783 KDa
SequenceString:
(DC)(DG)(DA)(DA)(DA)(DA)(DC)(DG)(DA)(DC) (DG)(DG)(DC)(DC)(DA)(DG)(DT)(DG)(DC)(DC) (DA)(DT)(DA)(DT)

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Macromolecule #4: DNA (5'-D(P*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP...

MacromoleculeName: DNA (5'-D(P*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.644536 KDa
SequenceString:
(DC)(DG)(DA)(DG)(DG)(DT)(DA)(DT)(DG)(DG) (DC)(DA)(DC)(DT)(DG)(DG)(DC)(DC)(DG)(DT) (DC)(DG)(DT)(DT)(DT)(DT)(DC)(DG)

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Macromolecule #5: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: DCP
Molecular weightTheoretical: 467.157 Da
Chemical component information

ChemComp-DCP:
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / Deoxycytidine triphosphate

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryoSPARC ab-initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 357312
FSC plot (resolution estimation)

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