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- EMDB-42842: A Mitochondrial Replication Complex: PolG/PolG2 Bound to DNA in c... -

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Basic information

Entry
Database: EMDB / ID: EMD-42842
TitleA Mitochondrial Replication Complex: PolG/PolG2 Bound to DNA in complex with the Single-Stranded Binding Protein (mtSSB)
Map data
Sample
  • Complex: The Mitchondrial DNA polymerase, catalytic (PolG) and accessory subunits (PolG2), bound to DNA in complex with the mitochondrial DNA single-stranded binding protein (mtSSB)
    • Protein or peptide: PolG, mitochondrial DNA polymerase catalytic subunit
    • Protein or peptide: PolG2, mitochondrial DNA polymerase gamma accessory subunit
    • Protein or peptide: Single-stranded DNA-binding protein, mitochondrial
    • DNA: DNA1
    • DNA: DNA2
    • DNA: DNA3
KeywordsDNA BINDING PROTEIN / DNA polymerase / mitochondrial DNA replication / DNA polymerase gamma / mitochondrial single-stranded binding protein / SSB / SSBP1 / DNA BINDING PROTEIN-DNA complex
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRiccio AA / Bouvette J / Borgnia MJ / Copeland WC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065078 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065080 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structures of the mitochondrial single-stranded DNA binding protein with DNA and DNA polymerase γ.
Authors: Amanda A Riccio / Jonathan Bouvette / Lars C Pedersen / Shruti Somai / Robert C Dutcher / Mario J Borgnia / William C Copeland /
Abstract: The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. ...The mitochondrial single-stranded DNA (ssDNA) binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA that could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Polγ, has been minimally explored. Here, we determined a 1.9-Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low-affinity site and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses a clinically relevant residue, R38, and a highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Polγ. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Polγ that mtSSB binds.
History
DepositionNov 16, 2023-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateOct 2, 2024-
Current statusOct 2, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42842.png

Downloads & links

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Map

FileDownload / File: emd_42842.map.gz / Format: CCP4 / Size: 699 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.)
0.85 Å/pix.
x 568 pix.
= 482.8 Å		0.85 Å/pix.
x 568 pix.
= 482.8 Å		0.85 Å/pix.
x 568 pix.
= 482.8 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.155
Minimum - Maximum-0.41639942 - 0.9384343
Average (Standard dev.)-0.0005505704 (±0.017197749)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions568568568
Spacing568568568
CellA=B=C: 482.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42842_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_42842_half_map_2.map
Projections & Slices
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Sample components

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Entire : The Mitchondrial DNA polymerase, catalytic (PolG) and accessory s...

EntireName: The Mitchondrial DNA polymerase, catalytic (PolG) and accessory subunits (PolG2), bound to DNA in complex with the mitochondrial DNA single-stranded binding protein (mtSSB)
Components
  • Complex: The Mitchondrial DNA polymerase, catalytic (PolG) and accessory subunits (PolG2), bound to DNA in complex with the mitochondrial DNA single-stranded binding protein (mtSSB)
    • Protein or peptide: PolG, mitochondrial DNA polymerase catalytic subunit
    • Protein or peptide: PolG2, mitochondrial DNA polymerase gamma accessory subunit
    • Protein or peptide: Single-stranded DNA-binding protein, mitochondrial
    • DNA: DNA1
    • DNA: DNA2
    • DNA: DNA3

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Supramolecule #1: The Mitchondrial DNA polymerase, catalytic (PolG) and accessory s...

SupramoleculeName: The Mitchondrial DNA polymerase, catalytic (PolG) and accessory subunits (PolG2), bound to DNA in complex with the mitochondrial DNA single-stranded binding protein (mtSSB)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: PolG, mitochondrial DNA polymerase catalytic subunit

MacromoleculeName: PolG, mitochondrial DNA polymerase catalytic subunit / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGGSHHHHHH GSRFMVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQL RHNPLDIQML SRGLHEQIFG QGGEMPGEAA VRRSVEHLQK H GLWGQPAV PLPDVELRLP PLYGDNLDQH FRLLAQKQSL PYLEAANLLL QA QLPPKPP AWAWAEGWTR ...String:
MGGSHHHHHH GSRFMVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQL RHNPLDIQML SRGLHEQIFG QGGEMPGEAA VRRSVEHLQK H GLWGQPAV PLPDVELRLP PLYGDNLDQH FRLLAQKQSL PYLEAANLLL QA QLPPKPP AWAWAEGWTR YGPEGEAVPV AIPEERALVF AVAVCLAEGT CPT LAVAIS PSAWYSWCSQ RLVEERYSWT SQLSPADLIP LEVPTGASSP TQRD WQEQL VVGHNVSFDR AHIREQYLIQ GSRMRFLDTM SMHMAISGLS SFQRS LWIA AKQGKHKVQP PTKQGQKSQR KARRGPAISS WDWLDISSVN SLAEVH RLY VGGPPLEKEP RELFVKGTMK DIRENFQDLM QYCAQDVWAT HEVFQQQ LP LFLERCPHPV TLAGMLEMGV SYLPVNQNWE RYLAEAQGTY EELQREMK K SLMDLANDAC QLLSGERYKE DPWLWDLEWD LQEFKQKKAK KVKKEPATA SKLPIEGAGA PGDPMDQEDL GPCSEEEEFQ QDVMARACLQ KLKGTTELLP KRPQHLPGH PGWYRKLCPR LDDPAWTPGP SLLSLQMRVT PKLMALTWDG F PLHYSERH GWGYLVPGRR DNLAKLPTGT TLESAGVVCP YRAIESLYRK HC LEQGKQQ LMPQEAGLAE EFLLTDNSAI WQTVEELDYL EVEAEAKMEN LRA AVPGQP LALTARGGPK DTQPSYHHGN GPYNDVDIPG CWFFKLPHKD GNSC NVGSP FAKDFLPKME DGTLQAGPGG ASGPRALEIN KMISFWRNAH KRISS QMVV WLPRSALPRA VIRHPDYDEE GLYGAILPQV VTAGTITRRA VEPTWL TAS NARPDRVGSE LKAMVQAPPG YTLVGADVDS QELWIAAVLG DAHFAGM HG CTAFGWMTLQ GRKSRGTDLH SKTATTVGIS REHAKIFNYG RIYGAGQP F AERLLMQFNH RLTQQEAAEK AQQMYAATKG LRWYRLSDEG EWLVRELNL PVDRTEGGWI SLQDLRKVQR ETARKSQWKK WEVVAERAWK GGTESEMFNK LESIATSDI PRTPVLGCCI SRALEPSAVQ EEFMTSRVNW VVQSSAVDYL H LMLVAMKW LFEEFAIDGR FCISIHDEVR YLVREEDRYR AALALQITNL LT RCMFAYK LGLNDLPQSV AFFSAVDIDR CLRKEVTMDC KTPSNPTGME RRY GIPQGE ALDIYQIIEL TKGSLEKRSQ PGP

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Macromolecule #2: PolG2, mitochondrial DNA polymerase gamma accessory subunit

MacromoleculeName: PolG2, mitochondrial DNA polymerase gamma accessory subunit
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: M A S R G S H H H H H H G A DA GQPELLTERS SPKGGHVKSH AELEGNGEHP EAP GSGEGS EALLEICQRR HFLSGSKQQL SRDSLLSGCH PGFGPLGVEL RKNLAAEWWT SVVV FREQV FPVDALHHKP GPLLPGDSAF RLVSAETLRE ILQDKELSKE ...String:
M A S R G S H H H H H H G A DA GQPELLTERS SPKGGHVKSH AELEGNGEHP EAP GSGEGS EALLEICQRR HFLSGSKQQL SRDSLLSGCH PGFGPLGVEL RKNLAAEWWT SVVV FREQV FPVDALHHKP GPLLPGDSAF RLVSAETLRE ILQDKELSKE QLVAFLENVL KTSGK LREN LLHGALEHYV NCLDLVNKRL PYGLAQIGVC FHPVFDTKQI RNGVKSIGEK TEASLV WFT PPRTSNQWLD FWLRHRLQWW RKFAMSPSNF SSSDCQDEEG RKGNKLYYNF PWGKELI ET LWNLGDHELL HMYPGNVSKL HGRDGRKNVV PCVLSVNGDL DRGMLAYLYD SFQLTENS F TRKKNLHRKV LKLHPCLAPI KVALDVGRGP TLELRQVCQG LFNELLENGI SVWPGYLET MQSSLEQLYS KYDEMSILFT VLVTETTLEN GLIHLRSRDT TMKEMMHISK LKDFLIKYIS SAKNV

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Macromolecule #3: Single-stranded DNA-binding protein, mitochondrial

MacromoleculeName: Single-stranded DNA-binding protein, mitochondrial / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString:
MGSSHHHHHH SSGENLYFQG SMESETTTSL VLERSLNRVH LLGRVGQDPV LRQVEGKNP VTIFSLATNE MWRSGDSEVY QLGDVSQKTT WHRISVFRPG L RDVAYQYV KKGSRIYLEG KIDYGEYMDK NNVRRQATTI IADNIIFLSD QT KEKE

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Macromolecule #4: DNA1

MacromoleculeName: DNA1 / type: dna / ID: 4 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
ATTTCGTACA TTACTGCCAG CCACCATGA ATATTGTACG GTACCATAAA TACTTGACCA CCTGTAGTAC ATAAAAACCC A

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Macromolecule #5: DNA2

MacromoleculeName: DNA2 / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
TGGGTTTTTA TGTACTACAG GTGGTCAAGT ATTTATGGTA CCGTACAATA

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Macromolecule #6: DNA3

MacromoleculeName: DNA3 / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
AGACACAGCA CTACATTGAG GTTGGTACGA GCAGTTAGGG AGAATTGAGC TCTAACGGAT CTGGCAGTA ATGTACGAAA T

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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

Concentration1.2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / 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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 61961
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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