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- EMDB-15385: FEN1 holoenzyme with a nicked DNA substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-15385
TitleFEN1 holoenzyme with a nicked DNA substrate
Map data
Sample
  • Complex: complex of FEN1 with PCNA and DNA
    • Complex: Proliferating cell nuclear antigen and Flap Endonuclease I
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Flap Endonuclease I
    • Complex: Oligo19ddC, Oligo13P and Oligo32
      • DNA: Oligo19ddC
      • DNA: Oligo13P
      • DNA: Oligo32
KeywordsDNA / Replication / Complex / Ligase / PCNA / Ligation / FEN1 / Flap Endonuclease I / Okazaki fragment maturation
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsBlair K / Tehseen M / Raducanu VS / Shahid T / Lancey C / Cruehet R / Hamdan S / De Biasio A
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing.
Authors: Kerry Blair / Muhammad Tehseen / Vlad-Stefan Raducanu / Taha Shahid / Claudia Lancey / Fahad Rashid / Ramon Crehuet / Samir M Hamdan / Alfredo De Biasio /
Abstract: During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present ...During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP) and DNA binding domain (PIP). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP is released from PCNA and only PIP is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.
History
DepositionJul 13, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15385.png

Downloads & links

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Map

FileDownload / File: emd_15385.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 216 pix.
= 180.36 Å		0.84 Å/pix.
x 216 pix.
= 180.36 Å		0.84 Å/pix.
x 216 pix.
= 180.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.835 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.031164812 - 0.042129107
Average (Standard dev.)0.00015845036 (±0.002693406)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 180.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15385_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15385_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex of FEN1 with PCNA and DNA

EntireName: complex of FEN1 with PCNA and DNA
Components
  • Complex: complex of FEN1 with PCNA and DNA
    • Complex: Proliferating cell nuclear antigen and Flap Endonuclease I
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
      • Protein or peptide: Flap Endonuclease I
    • Complex: Oligo19ddC, Oligo13P and Oligo32
      • DNA: Oligo19ddC
      • DNA: Oligo13P
      • DNA: Oligo32

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Supramolecule #1: complex of FEN1 with PCNA and DNA

SupramoleculeName: complex of FEN1 with PCNA and DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Proliferating cell nuclear antigen and Flap Endonuclease I

SupramoleculeName: Proliferating cell nuclear antigen and Flap Endonuclease I
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Oligo19ddC, Oligo13P and Oligo32

SupramoleculeName: Oligo19ddC, Oligo13P and Oligo32 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5-#7
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD ...String:
GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD GVKFSASGEL GNGNIKLSQT SNVDKEEEAV TIEMNEPVQL TFALRYLNFF TKATPLSSTV TLSMSADVPL VV EYKIADM GHLKYYLAPK IEDEEGS

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Macromolecule #2: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD ...String:
GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD GVKFSASGEL GNGNIKLSQT SNVDKEEEAV TIEMNEPVQL TFALRYLNFF TKATPLSSTV TLSMSADVPL VV EYKIADM GHLKYYLAPK IEDEEGS

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Macromolecule #3: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD ...String:
GPHMFEARLV QGSILKKVLE ALKDLINEAC WDISSSGVNL QSMDSSHVSL VQLTLRSEGF DTYRCDRNLA MGVNLTSMSK ILKCAGNED IITLRAEDNA DTLALVFEAP NQEKVSDYEM KLMDLDVEQL GIPEQEYSCV VKMPSGEFAR ICRDLSHIGD A VVISCAKD GVKFSASGEL GNGNIKLSQT SNVDKEEEAV TIEMNEPVQL TFALRYLNFF TKATPLSSTV TLSMSADVPL VV EYKIADM GHLKYYLAPK IEDEEGS

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Macromolecule #4: Flap Endonuclease I

MacromoleculeName: Flap Endonuclease I / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGIQGLAKLI ADVAPSAIRE NDIKSYFGRK VAIDASMSIY QFLIAVRQGG DVLQNEEGET TSHLMGMFYR TIRMMENGIK PVYVFDGKP PQLKSGELAK RSERRAEAEK QLQQAQAAGA EQEVEKFTKR LVKVTKQHND ECKHLLSLMG IPYLDAPSEA E ASCAALVK ...String:
MGIQGLAKLI ADVAPSAIRE NDIKSYFGRK VAIDASMSIY QFLIAVRQGG DVLQNEEGET TSHLMGMFYR TIRMMENGIK PVYVFDGKP PQLKSGELAK RSERRAEAEK QLQQAQAAGA EQEVEKFTKR LVKVTKQHND ECKHLLSLMG IPYLDAPSEA E ASCAALVK AGKVYAAATE DMACLTFGSP VLMRHLTASE AKKLPIQEFH LSRILQELGL NQEQFVDLCI LLGSDYCESI RG IGPKRAV DLIQKHKSIE EIVRRLDPNK YPVPENWLHK EAHQLFLEPE VLDPESVELK WSEPNEEELI KFMCGEKQFS EER IRSGVK RLSKSRQGST QGRLDDFFKV TGSLSSAKRK EPEPKGSTKK KAKTGAAGKF KRGK

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Macromolecule #5: Oligo19ddC

MacromoleculeName: Oligo19ddC / type: dna / ID: 5 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(DG)(DC)(DT)(DT)(DC)(DT)(DG)(DT)(DG)(DC) (DT)(DG)(DA)(DT)(DG)(DC)(DG)(DT)(DOC)

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Macromolecule #6: Oligo13P

MacromoleculeName: Oligo13P / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(PO4)(DG)(DT)(DC)(DG)(DG)(DA)(DC)(DT)(DG) (DA)(DA)(DC)(DC)

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Macromolecule #7: Oligo32

MacromoleculeName: Oligo32 / type: dna / ID: 7 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(DG)(DG)(DT)(DT)(DC)(DA)(DG)(DT)(DC)(DC) (DG)(DA)(DC)(DG)(DA)(DC)(DG)(DC)(DA)(DT) (DC)(DA)(DG)(DC)(DA)(DC)(DA)(DG)(DA) (DA)(DG)(DC)

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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: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4S4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
40.0 mMCH3CO2Kpotassium acetate
10.0 mMMgCl2magnesium chloride
0.5 mMC9H15O6Ptris(2-carboxyethyl)phosphine
0.1 mMC10H16N5O13P3ATP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec.
Details: The grid was coated with graphene oxide prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 18.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Startup modelType of model: OTHER / Details: Relion Ab initio model
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 118181
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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