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- EMDB-17815: Pyrococcus abyssi DNA polymerase D (PolD) in its editing mode bou... -

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Basic information

Entry
Database: EMDB / ID: EMD-17815
TitlePyrococcus abyssi DNA polymerase D (PolD) in its editing mode bound to a primer/template substrate containing a mismatch
Map data
Sample
  • Complex: Binary complex of PolD bound to PCNA and a primer/template duplex containing three consecutive mismatches
    • DNA: DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*GP*CP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')
    • Protein or peptide: DNA polymerase II small subunit
    • Protein or peptide: DP2
    • Protein or peptide: DNA polymerase sliding clampDNA clamp
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsPolymerase / DNA / Replication / PolD / Archaea / Editing / Proofreading / Exonuclease / Nuclease / DNA BINDING PROTEIN
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / DNA catabolic process / DNA polymerase processivity factor activity / regulation of DNA replication / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : ...DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA polymerase sliding clamp / DNA polymerase II small subunit
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBetancurt-Anzola L / Martinez-Carranza M / Zatopek KM / Gardner AF / Sauguet L
Funding support France, 1 items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR) France
CitationJournal: Nat Commun / Year: 2023
Title: Molecular basis for proofreading by the unique exonuclease domain of Family-D DNA polymerases.
Authors: Leonardo Betancurt-Anzola / Markel Martínez-Carranza / Marc Delarue / Kelly M Zatopek / Andrew F Gardner / Ludovic Sauguet /
Abstract: Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. ...Replicative DNA polymerases duplicate entire genomes at high fidelity. This feature is shared among the three domains of life and is facilitated by their dual polymerase and exonuclease activities. Family D replicative DNA polymerases (PolD), found exclusively in Archaea, contain an unusual RNA polymerase-like catalytic core, and a unique Mre11-like proofreading active site. Here, we present cryo-EM structures of PolD trapped in a proofreading mode, revealing an unanticipated correction mechanism that extends the repertoire of protein domains known to be involved in DNA proofreading. Based on our experimental structures, mutants of PolD were designed and their contribution to mismatch bypass and exonuclease kinetics was determined. This study sheds light on the convergent evolution of structurally distinct families of DNA polymerases, and the domain acquisition and exchange mechanism that occurred during the evolution of the replisome in the three domains of life.
History
DepositionJul 10, 2023-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17815.png

Downloads & links

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Map

FileDownload / File: emd_17815.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.2631608 - 1.9993773
Average (Standard dev.)-0.00020333761 (±0.038777266)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 344.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17815_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_17815_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_17815_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 : Binary complex of PolD bound to PCNA and a primer/template duplex...

EntireName: Binary complex of PolD bound to PCNA and a primer/template duplex containing three consecutive mismatches
Components
  • Complex: Binary complex of PolD bound to PCNA and a primer/template duplex containing three consecutive mismatches
    • DNA: DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*GP*CP*TP*TP*T)-3')
    • DNA: DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')
    • Protein or peptide: DNA polymerase II small subunit
    • Protein or peptide: DP2
    • Protein or peptide: DNA polymerase sliding clampDNA clamp
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Binary complex of PolD bound to PCNA and a primer/template duplex...

SupramoleculeName: Binary complex of PolD bound to PCNA and a primer/template duplex containing three consecutive mismatches
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #3-#4, #1, #5, #2
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)

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Macromolecule #1: DNA polymerase II small subunit

MacromoleculeName: DNA polymerase II small subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 74.009742 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGV FIIDGDLAYE FLQFLGLGVP QEIKESYIST GEEAEKTVES QETRASELEE GGVSQVSSGE LQELKEESPE I STTEEEIG ...String:
MGKHHHHSGH HHTGHHHHSG SHHHTSSSAS TGENLYFQGT GDGSDELVKA LERAGYLLTP SAYYLLVDHF KEGKFSLVEL VKFAKSKGV FIIDGDLAYE FLQFLGLGVP QEIKESYIST GEEAEKTVES QETRASELEE GGVSQVSSGE LQELKEESPE I STTEEEIG GLELVQSSIS TGSEVEYNNG ENGESVVVLD KYGYPILYAP EEIGEEKEYS KYEDVVIEWN PSVTPVQIEK NY EVKFDVR QVKLRPPKVK NGSGKEGEII VEAYASLFKS RLSKLKRILR ENPEISNVVD IGKLNYVSGD EEVTIIGLVN SKR ETNRGL IFEVEDKTGI VKVFLPKDSE DYREAFKVLP DAVVAFKGFY SKKGIFFANK FYLPDVPLYR KQKPPLEEKV YAIL ISDIH VGSREFCEKA FLKFLEWLNG HVESKEEEEI VSRVKYLIIA GDVVDGIGIY PGQYSDLVIP DIFDQYEALA NLLAN VPEH ITMFIGPGNA DAARPAIPQP EFYKEYAKPI YKLKNAIIIS NPAVIRLHGR DFLIAHGRGI EDVVSFVPGL THHKPG LPM VELLKMRHLA PTFGGKVPIA PDPEDLLVIE EVPDLVQMGH VHVYDAVVYR GVQLVNSATW QAQTEFQKMV NIVPTPA KV PVVDVESARV VKVLDFSGWC

UniProtKB: DNA polymerase II small subunit

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Macromolecule #2: DNA polymerase sliding clamp

MacromoleculeName: DNA polymerase sliding clamp / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 29.471764 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRGSHHHHHH GSMPFEIVFE GAKEFAQLIE TASRLIDEAA FKVTEEGISM RAMDPSRVVL IDLNLPASIF SKYEVDGEET IGVNMDHLK KVLKRGKAKE TLILRKGEEN FLEISLQGTA TRTFKLPLID VEEIEVDLPE LPFTAKVVIL GDVIKEAVKD A SLVSDSMK ...String:
MRGSHHHHHH GSMPFEIVFE GAKEFAQLIE TASRLIDEAA FKVTEEGISM RAMDPSRVVL IDLNLPASIF SKYEVDGEET IGVNMDHLK KVLKRGKAKE TLILRKGEEN FLEISLQGTA TRTFKLPLID VEEIEVDLPE LPFTAKVVIL GDVIKEAVKD A SLVSDSMK FIAKENEFTM RAEGETQEVE VKLTLEDEGL LDIEVQEETK SAYGISYLSD MVKGLGKADE VTIKFGNEMP MQ MEYYIRD EGRLIFLLAP RVEE

UniProtKB: DNA polymerase sliding clamp

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Macromolecule #5: DP2

MacromoleculeName: DP2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 144.418969 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKF GDLGSREKYA EQAVRTALAI LTEGIVSAPI EGIANVKIKR NTWADNSEYL ALYYAGPIRS SGGTAQALSV L VGDYVRRK ...String:
MELPKEMEEY FEMLQREIDK AYEIAKKARA QGKDPSLDVE IPQATDMAGR VESLVGPPGV AKRIRELVKE YGKEIAALKI VDEIIEGKF GDLGSREKYA EQAVRTALAI LTEGIVSAPI EGIANVKIKR NTWADNSEYL ALYYAGPIRS SGGTAQALSV L VGDYVRRK LGLDRFKPSE KHIERMVEEV DLYHRAVTRL QYHPSPEEVR LAMRNIPIEI TGEATDDVEV SHRDVPGVET NQ LRGGAIL VLAEGVLQKA KKLVKYIDKM GIEGWEWLKE FVEAKEKGEP KEEGKEESLA ESTLEETKVE VDMGFYYSLY QKF KEEIAP SDKYAKEVIG GRPLFSDPSK PGGFRLRYGR SRASGFATWG INPATMILVD EFLAIGTQLK TERPGKGAVV TPVT TIEGP IVKLKDGSVL RVDDYNLALK VREDVEEILY LGDAVIAFGD FVENNQTLLP ANYCEEWWIL EFVKALKEIY EVHLE PFTE NEEESIEEAS DYLEIDPEFL KEMLRDPLRV KPPVELAIHF SEVLGIPLHP YYTLYWNSVE PKDVEKLWRL LKNYAE IEW SNFRGIKFAK KIVISQEKLG DSKRTLELLG LPHTVRDGNV IVDYPWAAAL LTPLGNLNWE FMAKPLYATI DIINENN EI KLRDRGISWI GARMGRPEKA KERKMKPPVQ VLFPIGLAGG SSRDIKKAAE EGKVAEVEIA FFKCPKCGHV GPEHLCPN C GTRKELLWVC PRCNAEYPES QAEGYNYTCP KCNVKLRPYA KRKIRPSELL NRAMENVKVY GVDKLKGVMG MTSGWKMPE PLEKGLLRAK NDVYVFKDGT IRFDATDAPI THFRPREIGV SVEKLRELGY THDFEGKPLV SEDQIVELKP QDIILSKEAG RYLLKVAKF VDDLLEKFYG LPRFYNAEKM EDLIGHLVIG LAPHTSAGIV GRIIGFVDAL VGYAHPYFHA AKRRNCDGDE D AVMLLLDA LLNFSRYYLP EKRGGKMDAP LVITTRLDPR EVDSEVHNMD IVRYYPLEFY EATYELKSPK ELVGVIERVE DR LGKPEMY YGLKFTHDTD DIALGPKMSL YKQLGDMEEK VRRQLEVAKR IRAVDEHGVA EKILNSHLIP DLRGNLRSFT RQE FRCVKC NTKFRRPPLN GKCPVCGGKI VLTVSKGAIE KYLGTAKMLV TEYNVKNYTR QRICLTERDI DSLFENVFPE TQLT LIVNP NDICQRLVMA RTGEVNKSGL LENLSNGSKK TEKAEKAEKP RKKSDEKPKK KRVISLEEFF SRKSK

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Macromolecule #3: DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*GP*CP*TP*TP...

MacromoleculeName: DNA (5'-D(P*CP*CP*GP*GP*GP*CP*CP*GP*AP*GP*CP*CP*GP*TP*GP*CP*TP*TP*T)-3')
type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 5.519542 KDa
SequenceString:
(DC)(DG)(DC)(DC)(DG)(DG)(DG)(DC)(DC)(DG) (DA)(DG)(DC)(DC)(DG)(DT)(DG)(DC)

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Macromolecule #4: DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')

MacromoleculeName: DNA (5'-D(P*AP*GP*CP*AP*CP*GP*GP*CP*TP*CP*GP*GP*CP*CP*CP*GP*G)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pyrococcus abyssi GE5 (archaea)
Molecular weightTheoretical: 7.741958 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DC)(DG)(DT)(DG)(DA)(DA) (DC)(DG)(DG)(DC)(DT)(DC)(DG)(DG)(DC)(DC) (DC)(DG)(DG)(DC)(DG)

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6t8h

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 496901
FSC plot (resolution estimation)

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