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- EMDB-10401: Cryo-EM structure of the DNA-bound PolD-PCNA processive complex f... -

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Basic information

Entry
Database: EMDB / ID: EMD-10401
TitleCryo-EM structure of the DNA-bound PolD-PCNA processive complex from P. abyssi
Map dataPost processed and masked map of the PolD-PCNA-DNA from P. abyssi
Sample
  • Complex: DNA-bound PolD-PCNA processive complex from P. abyssi
    • Complex: PolD-PCNA processive complex
      • Protein or peptide: DNA polymerase sliding clampDNA clamp
      • Protein or peptide: DNA polymerase II small subunit
      • Protein or peptide: DP2 subunit of D-family DNA-polymerase
    • Complex: DNA
      • DNA: DNA primerPrimer (molecular biology)
      • DNA: DNA template
  • Ligand: FE (III) ION
  • Ligand: ZINC ION
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / 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 large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / DNA polymerase II small subunit, archaeal / DNA polymerase delta/II small subunit family / Intein splicing domain / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Hint (Hedgehog/Intein) domain N-terminal region / 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 / Hint domain superfamily / : / 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 / DNA polymerase II large subunit
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea) / synthetic construct (others) / Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsMadru C / Raia P / Hugonneau Beaufet I / Pehau-Arnaudet G / England P / Lindhal E / Delarue M / Carroni M / Sauguet L
Funding support France, Sweden, 3 items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0005-01 France
Swedish Research Council2017-04641 Sweden
Knut and Alice Wallenberg FoundationKAW 2015.0234 Sweden
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA.
Authors: Clément Madru / Ghislaine Henneke / Pierre Raia / Inès Hugonneau-Beaufet / Gérard Pehau-Arnaudet / Patrick England / Erik Lindahl / Marc Delarue / Marta Carroni / Ludovic Sauguet /
Abstract: Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced ...Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
History
DepositionOct 24, 2019-
Header (metadata) releaseMar 4, 2020-
Map releaseMar 4, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t8h
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10401.png

Downloads & links

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Map

FileDownload / File: emd_10401.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost processed and masked map of the PolD-PCNA-DNA from P. abyssi
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.061317734 - 0.10814589
Average (Standard dev.)0.00027422933 (±0.0027429883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z249.000249.000249.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0610.1080.000

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Supplemental data

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Mask #1

Fileemd_10401_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Post processed unmasked map of the PolD-PCNA-DNA from P. abyssi

Fileemd_10401_additional_1.map
AnnotationPost processed unmasked map of the PolD-PCNA-DNA from P. abyssi
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered map of the PolD-PCNA-DNA from P. abyssi

Fileemd_10401_additional_2.map
AnnotationUnfiltered map of the PolD-PCNA-DNA from P. abyssi
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the PolD-PCNA-DNA from P. abyssi

Fileemd_10401_half_map_1.map
AnnotationHalf map 2 of the PolD-PCNA-DNA from P. abyssi
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of the PolD-PCNA-DNA from P. abyssi

Fileemd_10401_half_map_2.map
AnnotationHalf map 1 of the PolD-PCNA-DNA from P. abyssi
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DNA-bound PolD-PCNA processive complex from P. abyssi

EntireName: DNA-bound PolD-PCNA processive complex from P. abyssi
Components
  • Complex: DNA-bound PolD-PCNA processive complex from P. abyssi
    • Complex: PolD-PCNA processive complex
      • Protein or peptide: DNA polymerase sliding clampDNA clamp
      • Protein or peptide: DNA polymerase II small subunit
      • Protein or peptide: DP2 subunit of D-family DNA-polymerase
    • Complex: DNA
      • DNA: DNA primerPrimer (molecular biology)
      • DNA: DNA template
  • Ligand: FE (III) ION
  • Ligand: ZINC ION

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Supramolecule #1: DNA-bound PolD-PCNA processive complex from P. abyssi

SupramoleculeName: DNA-bound PolD-PCNA processive complex from P. abyssi / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #4-#5
Molecular weightExperimental: 310 kDa/nm

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Supramolecule #2: PolD-PCNA processive complex

SupramoleculeName: PolD-PCNA processive complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3
Source (natural)Organism: Pyrococcus abyssi (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #4-#5
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: DNA polymerase sliding clamp

MacromoleculeName: DNA polymerase sliding clamp / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus abyssi (strain GE5 / Orsay) (archaea) / Strain: GE5 / Orsay
Molecular weightTheoretical: 29.471764 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

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Macromolecule #2: DNA polymerase II small subunit

MacromoleculeName: DNA polymerase II small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Pyrococcus abyssi (strain GE5 / Orsay) (archaea) / Strain: GE5 / Orsay
Molecular weightTheoretical: 69.408008 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDELVKALER AGYLLTPSAY YLLVDHFKEG KFSLVELVKF AKSKGVFIID GDLAYEFLQF LGLGVPQEIK ESYISTGEEA EKTVESQET RASELEEGGV SQVSSGELQE LKEESPEIST TEEEIGGLEL VQSSISTGSE VEYNNGENGE SVVVLDKYGY P ILYAPEEI ...String:
MDELVKALER AGYLLTPSAY YLLVDHFKEG KFSLVELVKF AKSKGVFIID GDLAYEFLQF LGLGVPQEIK ESYISTGEEA EKTVESQET RASELEEGGV SQVSSGELQE LKEESPEIST TEEEIGGLEL VQSSISTGSE VEYNNGENGE SVVVLDKYGY P ILYAPEEI GEEKEYSKYE DVVIEWNPSV TPVQIEKNYE VKFDVRQVKL RPPKVKNGSG KEGEIIVEAY ASLFKSRLSK LK RILRENP EISNVVDIGK LNYVSGDEEV TIIGLVNSKR ETNRGLIFEV EDKTGIVKVF LPKDSEDYRE AFKVLPDAVV AFK GFYSKK GIFFANKFYL PDVPLYRKQK PPLEEKVYAI LISDIHVGSR EFCEKAFLKF LEWLNGHVES KEEEEIVSRV KYLI IAGDV VDGIGIYPGQ YSDLVIPDIF DQYEALANLL ANVPEHITMF IGPGNADAAR PAIPQPEFYK EYAKPIYKLK NAIII SNPA VIRLHGRDFL IAHGRGIEDV VSFVPGLTHH KPGLPMVELL KMRHLAPTFG GKVPIAPDPE DLLVIEEVPD LVQMGH VHV YDAVVYRGVQ LVNSATWQAQ TEFQKMVNIV PTPAKVPVVD VESARVVKVL DFSGWC

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Macromolecule #3: DP2 subunit of D-family DNA-polymerase

MacromoleculeName: DP2 subunit of D-family DNA-polymerase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Pyrococcus abyssi (archaea)
Molecular weightTheoretical: 144.762188 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GTGDGSELPK EMEEYFEMLQ REIDKAYEIA KKARAQGKDP SLDVEIPQAT DMAGRVESLV GPPGVAKRIR ELVKEYGKEI AALKIVDEI IEGKFGDLGS REKYAEQAVR TALAILTEGI VSAPIEGIAN VKIKRNTWAD NSEYLALYYA GPIRSSGGTA Q ALSVLVGD ...String:
GTGDGSELPK EMEEYFEMLQ REIDKAYEIA KKARAQGKDP SLDVEIPQAT DMAGRVESLV GPPGVAKRIR ELVKEYGKEI AALKIVDEI IEGKFGDLGS REKYAEQAVR TALAILTEGI VSAPIEGIAN VKIKRNTWAD NSEYLALYYA GPIRSSGGTA Q ALSVLVGD YVRRKLGLDR FKPSEKHIER MVEEVDLYHR AVTRLQYHPS PEEVRLAMRN IPIEITGEAT DDVEVSHRDV PG VETNQLR GGAILVLAEG VLQKAKKLVK YIDKMGIEGW EWLKEFVEAK EKGEPKEEGK EESLAESTLE ETKVEVDMGF YYS LYQKFK EEIAPSDKYA KEVIGGRPLF SDPSKPGGFR LRYGRSRASG FATWGINPAT MILVDEFLAI GTQLKTERPG KGAV VTPVT TIEGPIVKLK DGSVLRVDDY NLALKVREDV EEILYLGDAV IAFGDFVENN QTLLPANYCE EWWILEFVKA LKEIY EVHL EPFTENEEES IEEASDYLEI DPEFLKEMLR DPLRVKPPVE LAIHFSEVLG IPLHPYYTLY WNSVEPKDVE KLWRLL KNY AEIEWSNFRG IKFAKKIVIS QEKLGDSKRT LELLGLPHTV RDGNVIVDYP WAAALLTPLG NLNWEFMAKP LYATIDI IN ENNEIKLRDR GISWIGARMG RPEKAKERKM KPPVQVLFPI GLAGGSSRDI KKAAEEGKVA EVEIAFFKCP KCGHVGPE H LCPNCGTRKE LLWVCPRCNA EYPESQAEGY NYTCPKCNVK LRPYAKRKIR PSELLNRAME NVKVYGVDKL KGVMGMTSG WKMPEPLEKG LLRAKNDVYV FKDGTIRFDA TDAPITHFRP REIGVSVEKL RELGYTHDFE GKPLVSEDQI VELKPQDIIL SKEAGRYLL KVAKFVDDLL EKFYGLPRFY NAEKMEDLIG HLVIGLAPHT SAGIVGRIIG FVDALVGYAH PYFHAAKRRN C DGDEDAVM LLLDALLNFS RYYLPEKRGG KMDAPLVITT RLDPREVDSE VHNMDIVRYY PLEFYEATYE LKSPKELVGV IE RVEDRLG KPEMYYGLKF THDTDDIALG PKMSLYKQLG DMEEKVRRQL EVAKRIRAVD EHGVAEKILN SHLIPDLRGN LRS FTRQEF RCVKCNTKFR RPPLNGKCPV CGGKIVLTVS KGAIEKYLGT AKMLVTEYNV KNYTRQRICL TERDIDSLFE NVFP ETQLT LIVNPNDICQ RLVMARTGEV NKSGLLENLS NGSKKTEKAE KAEKPRKKSD EKPKKKRVIS LEEFFSRKSK

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Macromolecule #4: DNA primer

MacromoleculeName: DNA primer / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
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 #5: DNA template

MacromoleculeName: DNA template / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.717934 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DC)(DG)(DT)(DG)(DC)(DA) (DC)(DG)(DG)(DC)(DT)(DC)(DG)(DG)(DC)(DC) (DC)(DG)(DG)(DC)(DG)

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Macromolecule #6: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 4 / 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

Concentration0.1 mg/mL
BufferpH: 8
GridModel: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 5mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: 0.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 4602 / Average exposure time: 5.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Indicated in the M&M
CTF correctionSoftware - Name: Gctf
Startup modelType of model: NONE / Details: Ab-initio model generated using SGD
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 149200
FSC plot (resolution estimation)

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