+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14078 | |||||||||
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Title | human Lig1-DNA-PCNA complex reconstituted in absence of ATP | |||||||||
Map data | ||||||||||
Sample |
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Keywords | DNA / Replication / Complex / Ligase / PCNA / Ligation / Okazaki fragment maturation | |||||||||
Function / homology | Function and homology information Okazaki fragment processing involved in mitotic DNA replication / Regulation of MITF-M-dependent genes involved in DNA damage repair and senescence / DNA ligase activity / DNA ligase (ATP) / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / DNA ligase (ATP) activity / purine-specific mismatch base pair DNA N-glycosylase activity ...Okazaki fragment processing involved in mitotic DNA replication / Regulation of MITF-M-dependent genes involved in DNA damage repair and senescence / DNA ligase activity / DNA ligase (ATP) / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / DNA ligase (ATP) activity / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / DNA ligation / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Polymerase switching on the C-strand of the telomere / replisome / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / lagging strand elongation / response to L-glutamate / histone acetyltransferase binding / DNA biosynthetic process / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / response to dexamethasone / replication fork processing / Early Phase of HIV Life Cycle / nuclear replication fork / SUMOylation of DNA replication proteins / POLB-Dependent Long Patch Base Excision Repair / PCNA-Dependent Long Patch Base Excision Repair / anatomical structure morphogenesis / estrous cycle / mismatch repair / translesion synthesis / response to cadmium ion / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / epithelial cell differentiation / base-excision repair, gap-filling / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / replication fork / male germ cell nucleus / liver regeneration / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / base-excision repair / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / DNA recombination / damaged DNA binding / chromosome, telomeric region / nuclear body / cell division / intracellular membrane-bounded organelle / DNA repair / centrosome / chromatin binding / protein-containing complex binding / chromatin / negative regulation of transcription by RNA polymerase II / enzyme binding / mitochondrion / DNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.58 Å | |||||||||
Authors | Blair K / Tehseen M / Raducanu VS / Shahid T / Lancey C / Cruehet R / Hamdan S / De Biasio A | |||||||||
Funding support | United Kingdom, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14078.map.gz | 12.1 MB | EMDB map data format | |
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Header (meta data) | emd-14078-v30.xml emd-14078.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_14078_fsc.xml | 9.3 KB | Display | FSC data file |
Images | emd_14078.png | 68 KB | ||
Filedesc metadata | emd-14078.cif.gz | 7.3 KB | ||
Others | emd_14078_half_map_1.map.gz emd_14078_half_map_2.map.gz | 50.7 MB 50.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14078 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14078 | HTTPS FTP |
-Validation report
Summary document | emd_14078_validation.pdf.gz | 898.3 KB | Display | EMDB validaton report |
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Full document | emd_14078_full_validation.pdf.gz | 897.8 KB | Display | |
Data in XML | emd_14078_validation.xml.gz | 15.2 KB | Display | |
Data in CIF | emd_14078_validation.cif.gz | 19.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14078 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14078 | HTTPS FTP |
-Related structure data
Related structure data | 7qnzMC 7qo1C 8b8tC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_14078.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.835 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_14078_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_14078_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Human Ligase holoenzyme without ATP present
Entire | Name: Human Ligase holoenzyme without ATP present |
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Components |
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-Supramolecule #1: Human Ligase holoenzyme without ATP present
Supramolecule | Name: Human Ligase holoenzyme without ATP present / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3, #5 |
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Source (natural) | Organism: Homo sapiens (human) |
-Macromolecule #1: DNA ligase 1
Macromolecule | Name: DNA ligase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA ligase (ATP) |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 101.877102 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MQRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSESD SPVKRPGRKA ARVLGSEGEE EDEALSPAKG QKPALDCSQ VSPPRPATSP ENNASLSDTS PMDSSPSGIP KRRTARKQLP KRTIQEVLEE QSEDEDREAK RKKEEEEEET P KESLTEAE ...String: MQRSIMSFFH PKKEGKAKKP EKEASNSSRE TEPPPKAALK EWNGVVSESD SPVKRPGRKA ARVLGSEGEE EDEALSPAKG QKPALDCSQ VSPPRPATSP ENNASLSDTS PMDSSPSGIP KRRTARKQLP KRTIQEVLEE QSEDEDREAK RKKEEEEEET P KESLTEAE VATEKEGEDG DQPTTPPKPL KTSKAETPTE SVSEPEVATK QELQEEEEQT KPPRRAPKTL SSFFTPRKPA VK KEVKEEE PGAPGKEGAA EGPLDPSGYN PAKNNYHPVE DACWKPGQKV PYLAVARTFE KIEEVSARLR MVETLSNLLR SVV ALSPPD LLPVLYLSLN HLGPPQQGLE LGVGDGVLLK AVAQATGRQL ESVRAEAAEK GDVGLVAENS RSTQRLMLPP PPLT ASGVF SKFRDIARLT GSASTAKKID IIKGLFVACR HSEARFIARS LSGRLRLGLA EQSVLAALSQ AVSLTPPGQE FPPAM VDAG KGKTAEARKT WLEEQGMILK QTFCEVPDLD RIIPVLLEHG LERLPEHCKL SPGIPLKPML AHPTRGISEV LKRFEE AAF TCEYKYDGQR AQIHALEGGE VKIFSRNQED NTGKYPDIIS RIPKIKLPSV TSFILDTEAV AWDREKKQIQ PFQVLTT RK RKEVDASEIQ VQVCLYAFDL IYLNGESLVR EPLSRRRQLL RENFVETEGE FVFATSLDTK DIEQIAEFLE QSVKDSCE G LMVKTLDVDA TYEIAKRSHN WLKLKKDYLD GVGDTLDLVV IGAYLGRGKR AGRYGGFLLA SYDEDSEELQ AICKLGTGF SDEELEEHHQ SLKALVLPSP RPYVRIDGAV IPDHWLDPSA VWEVKCADLS LSPIYPAARG LVDSDKGISL RFPRFIRVRE DKQPEQATT SAQVACLYRK QSQIQNQQGE DSGSDPEDTY UniProtKB: DNA ligase 1 |
-Macromolecule #2: Proliferating cell nuclear antigen
Macromolecule | Name: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 29.088061 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 UniProtKB: Proliferating cell nuclear antigen |
-Macromolecule #3: Oligo19ddC
Macromolecule | Name: Oligo19ddC / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.802744 KDa |
Sequence | String: (DG)(DC)(DT)(DT)(DC)(DT)(DG)(DT)(DG)(DC) (DT)(DG)(DA)(DT)(DG)(DC)(DG)(DT)(DOC) |
-Macromolecule #4: Oligo13P
Macromolecule | Name: Oligo13P / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 3.976599 KDa |
Sequence | String: (DG)(DT)(DC)(DG)(DG)(DA)(DC)(DT)(DG)(DA) (DA)(DC)(DC) |
-Macromolecule #5: Oligo32
Macromolecule | Name: Oligo32 / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 9.845345 KDa |
Sequence | String: (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) |
-Macromolecule #6: ADENOSINE MONOPHOSPHATE
Macromolecule | Name: ADENOSINE MONOPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: AMP |
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Molecular weight | Theoretical: 347.221 Da |
Chemical component information | ChemComp-AMP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: 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. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 77.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2966 / Average exposure time: 2.0 sec. / Average electron dose: 18.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | PDB-7qnz: |