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- EMDB-45196: Cryo-EM structure of the Strand displacement Complex (III) of Yea... -

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Entry
Database: EMDB / ID: EMD-45196
TitleCryo-EM structure of the Strand displacement Complex (III) of Yeast Mitochondrial DNA polymerase Gamma (MIP1) with downstream DNA
Map dataAnisotropic sharpened map of the original map
Sample
  • Complex: Cryo-EM map of the Strand displacement Complex (III) of Yeast Mitochondrial DNA polymerase Gamma (MIP1)
    • Protein or peptide: DNA polymerase gamma
    • DNA: Non-Template DNA
    • DNA: Primer DNA
    • DNA: Template DNA
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE
KeywordsMitochondrial DNA Polymerase Gamma / Strand displacement complex / MIP1 / REPLICATION / Helicase independent DNA polymerase / Transferase-DNA complex
Function / homology:
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsNayak AR / Sokolova VO / Sillamaa S / Sedmen J / Temiakov D / Zamudio-Ochoa A
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: Nat Commun / Year: 2025
Title: Structural basis for intrinsic strand displacement activity of mitochondrial DNA polymerase.
Authors: Ashok R Nayak / Viktoriia Sokolova / Sirelin Sillamaa / Karl Herbine / Juhan Sedman / Dmitry Temiakov /
Abstract: Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA ...Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA polymerase γ relies on the replicative helicase TWINKLE, whereas the yeast ortholog, Mip1, can unwind DNA independently. Using Mip1 as a model, we present a series of high-resolution cryo-EM structures that capture the process of DNA strand displacement. Our data reveal previously unidentified structural elements that facilitate the unwinding of the downstream DNA duplex. Yeast cells harboring Mip1 variants defective in strand displacement exhibit impaired oxidative phosphorylation and loss of mtDNA, corroborating the structural observations. This study provides a molecular basis for the intrinsic strand displacement activity of Mip1 and illuminates the distinct unwinding mechanisms utilized by Pol A family DNA polymerases.
History
DepositionJun 5, 2024-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45196.png

Downloads & links

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Map

FileDownload / File: emd_45196.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAnisotropic sharpened map of the original map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.77 Å/pix.
x 336 pix.
= 257.141 Å		0.77 Å/pix.
x 336 pix.
= 257.141 Å		0.77 Å/pix.
x 336 pix.
= 257.141 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7653 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.20782118 - 0.5319803
Average (Standard dev.)-0.000038506685 (±0.011386124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 257.14078 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEM sharpened map

Fileemd_45196_additional_1.map
AnnotationDeepEM sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Original unsharpened map

Fileemd_45196_additional_2.map
AnnotationOriginal unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_45196_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_45196_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM map of the Strand displacement Complex (III) of Yeast Mit...

EntireName: Cryo-EM map of the Strand displacement Complex (III) of Yeast Mitochondrial DNA polymerase Gamma (MIP1)
Components
  • Complex: Cryo-EM map of the Strand displacement Complex (III) of Yeast Mitochondrial DNA polymerase Gamma (MIP1)
    • Protein or peptide: DNA polymerase gamma
    • DNA: Non-Template DNA
    • DNA: Primer DNA
    • DNA: Template DNA
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

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Supramolecule #1: Cryo-EM map of the Strand displacement Complex (III) of Yeast Mit...

SupramoleculeName: Cryo-EM map of the Strand displacement Complex (III) of Yeast Mitochondrial DNA polymerase Gamma (MIP1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Yeast Mitochondrial DNA polymerase Gamma (MIP1)wild-type assembled on a primer-template and a downstream non-template strand with incoming nucleotide, dATP bound to the polymerase site.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 142 KDa

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Macromolecule #1: DNA polymerase gamma

MacromoleculeName: DNA polymerase gamma / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 141.745438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASTKKNTAE APRINPVGIQ YLGESLQRQV FGSCGGKDEV EQSDKLMELS KKSLKDHGLW GKKTLITDPI SFPLPPLQGR SLDEHFQKI GRFNSEPYKS FCEDKFTEMV ARPAEWLRKP GWVKYVPGMA PVEVAYPDEE LVVFDVETLY NVSDYPTLAT A LSSTAWYL ...String:
MASTKKNTAE APRINPVGIQ YLGESLQRQV FGSCGGKDEV EQSDKLMELS KKSLKDHGLW GKKTLITDPI SFPLPPLQGR SLDEHFQKI GRFNSEPYKS FCEDKFTEMV ARPAEWLRKP GWVKYVPGMA PVEVAYPDEE LVVFDVETLY NVSDYPTLAT A LSSTAWYL WCSPFICGGD DPAALIPLNT LNKEQVVIGH NVAYDRARVL EEYNFRDSKA FFLDTQSLHI ASFGLCSRQR PM FMKNNKK KEAEVESEVH PEISIEDYDD PWLNVSALNS LKDVAKFHCK IDLDKTDRDF FASTDKSTII ENFQKLVNYC ATD VTATSQ VFDKIFPVFL KKCPHPVSFA GLKSLSKCIL PTKLNDWNDY LNSSESLYQQ SKVQIESKIV QIIKDIALLK DKPD FYLKD PWLSQLDWTT KPLRLTKKGV PAKCQKLPGF PEWYRQLFPS KDTVEPKITI KSRIIPILFK LSWENSPVIW SKESG WCFN VPHEQVETYK AKNYVLADSV SQEEEEIRMN NLGLQCTGVL FKVPHPNGPT FNCTNLLTKS YNHFFEKGVL KSESEL AHQ ALQINSSGSY WMSARERIQS QFVVPNCKFP NEFQSLSAKS SLNNEKTNDL AIIIPKIVPM GTITRRAVEN TWLTASN AK ANRIGSELKT QVKAPPGYCF VGADVDSEEL WIASLVGDSI FNVHGGTAIG WMCLEGTKNE GTDLHTKTAQ ILGCSRNE A KIFNYGRIYG AGAKFASQLL KRFNPSLTDE ETKKIANKLY ENTKGKTKRS KLFKKFWYGG SESILFNKLE SIAEQETPK TPVLGCGITY SLMKKNLRAN SFLPSRINWA IQSSGVDYLH LLCCSMEYII KKYNLEARLC ISIHDEIRFL VSEKDKYRAA MALQISNIW TRAMFCQQMG INELPQNCAF FSQVDIDSVI RKEVNMDCIT PSNKTAIPHG EALDINQLLD KPNSKLGKPS L DIDSKVSQ YAYNYREPVF EEYNKSYTPE FLKYFLAMQV QSDKRDVNRL EDEYLRECTS KEYARDGNTA EYSLLDYIKD VE KGKRTKV RIMGSNFLDG TKNAKADQRI RLPVNMPDYP TLHKIANDSA IPEKQLLENR RKKENRIDDE NKKKLTRKKN TTP MERKYK RVYGGRKAFE AFYECANKPL DYTLETEKQF FNIPIDGVID DVLNDKSNYK KKPSQARTAS SSPIRKTAKA VHSK KLPAR KSSTTNRNLV ELERDITISR EYKLAAALEH HHHHH

UniProtKB: UNIPROTKB: A0A8H4BW69

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Macromolecule #2: Non-Template DNA

MacromoleculeName: Non-Template DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.078555 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DG)(DG)(DT)(DA)(DG)(DT) (DC)(DG)(DT)(DG)(DA)(DC)(DT)(DC)(DG)(DA) (DC)(DC)(DG)

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Macromolecule #3: Primer DNA

MacromoleculeName: Primer DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.17261 KDa
SequenceString:
(DG)(DA)(DA)(DG)(DA)(DC)(DA)(DG)(DT)(DC) (DT)(DG)(DC)(DG)(DG)(DC)(DG)(DC)(DG)(DC) (DG)(DG)(DG)

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Macromolecule #4: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.854218 KDa
SequenceString:
(DC)(DG)(DG)(DT)(DC)(DG)(DA)(DG)(DA)(DG) (DT)(DC)(DA)(DC)(DG)(DA)(DC)(DT)(DA)(DC) (DC)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DC) (DG)(DC)(DA)(DG)(DA)(DC)(DT)(DG)(DT)(DC) (DT) (DT)(DC)

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

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

MacromoleculeName: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: 2DT
Molecular weightTheoretical: 306.209 Da
Chemical component information

ChemComp-2DT:
3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

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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

Concentration0.57 mg/mL
BufferpH: 7.9 / Details: 10 mM Tris-HCL, 100 mM NaCL, 10 mM DTT, 5 mM MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsA 4 uM strand displacement complex complex was assembled with wild-type MIP1 and a DNA scaffold with primer, template, and non-template strands, mixed in equal millimolar ratio. The primer strand was extended by three nucleotides with 1 mM dGTP and chain terminated with 1 mM di-deoxy TTP. 1 mM dATP was added before plunge freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 10 eV
SoftwareName: EPU (ver. 3.7)
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 2 / Number real images: 14819 / Average electron dose: 60.0 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: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFalcon 4i
Particle selectionNumber selected: 9600000
Startup modelType of model: NONE / Details: Ab -initio reconstruction in cryoSPARC 4.4.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 137107
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 3
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1-f1


Chain - Residue range: 1-1254 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
SoftwareName: Coot (ver. 0.9.8.5)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9c53:
Cryo-EM structure of the Strand displacement Complex (II) of Yeast Mitochondrial DNA polymerase Gamma (MIP1) with downstream DNA

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