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- EMDB-27154: Human mitochondrial DNA polymerase gamma ternary complex with GC ... -

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Basic information

Entry
Database: EMDB / ID: EMD-27154
TitleHuman mitochondrial DNA polymerase gamma ternary complex with GC basepair
Map data
Sample
  • Complex: Human mitochondrial DNA polymerase gamma ternary complex with GC 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*C)-3')
    • DNA: DNA (25-MER)
  • Ligand: CALCIUM ION
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE
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 / base-excision repair / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding / protease 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
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. / Anticodon-binding domain superfamily / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / 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: 2.46 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)AI134611 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
DepositionMay 31, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27154.png

Downloads & links

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Map

FileDownload / File: emd_27154.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å		0.85 Å/pix.
x 320 pix.
= 272. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0
Minimum - Maximum0.0 - 16.607804999999999
Average (Standard dev.)0.32522085 (±0.6313453)
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_27154_additional_1.map
AnnotationCryoSPARC unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Half map: #2

Fileemd_27154_half_map_1.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27154_half_map_2.map
Projections & Slices
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Sample components

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

EntireName: Human mitochondrial DNA polymerase gamma ternary complex with GC basepair
Components
  • Complex: Human mitochondrial DNA polymerase gamma ternary complex with GC 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*C)-3')
    • DNA: DNA (25-MER)
  • Ligand: CALCIUM ION
  • Ligand: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

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

SupramoleculeName: Human mitochondrial DNA polymerase gamma ternary complex with GC 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: 140.559547 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 QPGPHHHHHH

UniProtKB: DNA polymerase subunit gamma-1

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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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.819867 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 VHHHHHH

UniProtKB: DNA polymerase subunit gamma-2

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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*C)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.357771 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)(DC)

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Macromolecule #4: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / 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: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: DCP
Molecular weightTheoretical: 467.157 Da
Chemical component information

ChemComp-DCP:
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE

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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
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
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
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 4494991
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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