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- EMDB-77235: Focused map (Accessory Subunit) of the Strand-Displacement Comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-77235
TitleFocused map (Accessory Subunit) of the Strand-Displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Map data
Sample
  • Complex: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
KeywordsMitochondrial DNA Polymerase Gamma / PolG / DNA strand displacement / REPLICATION
Function / homology
Function and homology information


gamma DNA polymerase complex / positive regulation of DNA-directed DNA polymerase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / DNA polymerase processivity factor activity / mitochondrial nucleoid / DNA polymerase binding / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding ...gamma DNA polymerase complex / positive regulation of DNA-directed DNA polymerase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / DNA polymerase processivity factor activity / mitochondrial nucleoid / DNA polymerase binding / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding / DNA-directed DNA polymerase activity / mitochondrial matrix / mitochondrion / identical protein binding
Similarity search - Function
POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / Anticodon-binding domain superfamily / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL)
Similarity search - Domain/homology
DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsNayak AR / Sokolova VO / Temiakov D
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM131832 United States
CitationJournal: Nucleic Acids Res / Year: 2026
Title: Molecular and structural basis for replication initiation and strand separation by human mitochondrial DNA polymerase γ.
Authors: Viktoriia Sokolova / Gina Buchel / Sarah Strock / Ashok R Nayak / Dmitry Temiakov /
Abstract: Defects in human mitochondrial DNA (mtDNA) replication can lead to somatic mutations associated with a range of devastating mitochondrial diseases. However, the molecular mechanisms governing the ...Defects in human mitochondrial DNA (mtDNA) replication can lead to somatic mutations associated with a range of devastating mitochondrial diseases. However, the molecular mechanisms governing the earliest steps of mtDNA replication and their fidelity remain poorly understood. Here, we found that DNA polymerase gamma (Polγ) forms stable complexes with RNA-DNA primer-template substrates, exhibiting greater stability and lower misincorporation than on DNA-primed substrates. Structural analysis revealed that Polγ interacts with the 2'-OH groups of ribose within the first four nucleotides of the primer, explaining the stability of complexes that utilize RNA primers. Although Polγ requires TWINKLE to extend RNA primers, its intrinsic strand-displacement activity allows it to extend DNA primers independently. Structural data further show that the strand-separation mechanism in human Polγ is distinct from that of its yeast paralog, Mip1, and involves previously unresolved elements-the catcher and a GP loop in the exonuclease domain-that support intrinsic strand-displacement synthesis by Polγ. Structure-guided mutagenesis of elements involved in strand separation supports these structural observations. Together, our study provides mechanistic insight into mtDNA replication initiation and strand separation and has implications for understanding the molecular basis of mitochondrial disease.
History
DepositionMay 19, 2026-
Header (metadata) releaseOct 7, 2026-
Map releaseOct 7, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_77235.png

Downloads & links

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Map

FileDownload / File: emd_77235.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 324 pix.
= 277.214 Å		0.86 Å/pix.
x 324 pix.
= 277.214 Å		0.86 Å/pix.
x 324 pix.
= 277.214 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8556 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0244
Minimum - Maximum-0.07544202 - 0.18456466
Average (Standard dev.)0.00021994078 (±0.003406044)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 277.2144 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_77235_msk_1.map
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Additional map: #1

Fileemd_77235_additional_1.map
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Half map: #1

Fileemd_77235_half_map_1.map
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Half map: #2

Fileemd_77235_half_map_2.map
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Sample components

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Entire : Strand displacement Complex of Human Mitochondrial DNA Polymerase...

EntireName: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Components
  • Complex: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial

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Supramolecule #1: Strand displacement Complex of Human Mitochondrial DNA Polymerase...

SupramoleculeName: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on a 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt 5' ...Details: Strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on a 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt 5' non-template flap ss-DNA. The primer terminus was extended by four nucleotides (three dG followed by a chain-terminating ddT), thereby promoting coordinated displacement of four non-template nucleotides from the downstream DNA duplex. The ternary catalytic complex is stabilized by a bound incoming nucleotide (dATP) at the polymerase active site in the presence of 5 mM MgCl2.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 362 KDa

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Macromolecule #1: DNA polymerase subunit gamma-2, mitochondrial

MacromoleculeName: DNA polymerase subunit gamma-2, mitochondrial / type: protein_or_peptide / ID: 1 / Details: Accessory subunit / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

UniProtKB: DNA polymerase subunit gamma-2

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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

Concentration0.52 mg/mL
BufferpH: 7.9
Details: 10 mM Tris-Hcl pH 7.9, 100 mM Nacl, 10 mM DTT, and 5 mM MgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details2 uM strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on an equimolar 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt single-stranded non-template flap. The primer was extended by four nucleotides; three successive dG at 0.2 mM, followed by incorporation of a chain-terminating ddT at 0.1 mM), thereby driving coordinated displacement of four nucleotides from the downstream DNA duplex. The resulting ternary catalytic complex was further stabilized by binding of an incoming nucleotide (1 mM dATP) at the polymerase active site in the presence of 5 mM MgCl2.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
SoftwareName: Latitude
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7236 / Average exposure time: 2.6 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5827907
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6) / Number images used: 395220
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.6)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9pck


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: Coot (ver. 0.9.8.5)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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