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- EMDB-10082: Human polymerase delta holoenzyme Conformer 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-10082
TitleHuman polymerase delta holoenzyme Conformer 3
Map data
Sample
  • Complex: PolD holoenzyme
    • Complex: Human polymerase delta
      • Protein or peptide: DNA polymerase delta catalytic subunit
      • Protein or peptide: DNA polymerase delta subunit 2
      • Protein or peptide: DNA polymerase delta subunit 3
      • Protein or peptide: DNA polymerase delta subunit 4
    • Complex: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
    • Complex: DNA
      • DNA: DNA primer
      • DNA: DNA template
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
KeywordsProtein / REPLICATION
Function / homology
Function and homology information


delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / nucleotide-excision repair complex / zeta DNA polymerase complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / Cytosolic iron-sulfur cluster assembly / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity ...delta DNA polymerase complex / DNA synthesis involved in UV-damage excision repair / nucleotide-excision repair complex / zeta DNA polymerase complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / Cytosolic iron-sulfur cluster assembly / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity / MutLalpha complex binding / nuclear lamina / Polymerase switching / nucleotide-excision repair, DNA gap filling / Telomere C-strand (Lagging Strand) Synthesis / 3'-5'-DNA exonuclease activity / Processive synthesis on the lagging strand / PCNA complex / DNA replication proofreading / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Removal of the Flap Intermediate from the C-strand / Transcription of E2F targets under negative control by DREAM complex / replisome / aggresome / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / response to L-glutamate / DNA strand elongation involved in DNA replication / histone acetyltransferase binding / error-free translesion synthesis / DNA synthesis involved in DNA repair / DNA biosynthetic process / leading strand elongation / DNA polymerase processivity factor activity / G1/S-Specific Transcription / response to dexamethasone / replication fork processing / nuclear replication fork / SUMOylation of DNA replication proteins / PCNA-Dependent Long Patch Base Excision Repair / estrous cycle / fatty acid homeostasis / mismatch repair / translesion synthesis / error-prone translesion synthesis / response to cadmium ion / DNA polymerase binding / cyclin-dependent protein kinase holoenzyme complex / response to UV / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of endothelial cell proliferation / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / male germ cell nucleus / replication fork / nuclear estrogen receptor binding / liver regeneration / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / receptor tyrosine kinase binding / DNA-templated DNA replication / 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 / protein-macromolecule adaptor activity / 4 iron, 4 sulfur cluster binding / DNA replication / damaged DNA binding / DNA-directed DNA polymerase / chromosome, telomeric region / DNA-directed DNA polymerase activity / nuclear body / DNA repair / nucleotide binding / centrosome / chromatin binding / protein-containing complex binding / chromatin / negative regulation of transcription by RNA polymerase II / enzyme binding / DNA binding / extracellular exosome
Similarity search - Function
DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta ...DNA polymerase delta, subunit 4 / DNA polymerase delta, subunit 4 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 3 superfamily / DNA polymerase subunit Cdc27 / DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase delta/II small subunit family / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Proliferating cell nuclear antigen signature 2. / : / 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 family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / : / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Proliferating cell nuclear antigen / DNA polymerase delta catalytic subunit / DNA polymerase delta subunit 2 / DNA polymerase delta subunit 3 / DNA polymerase delta subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsLancey C / Hamdan SM / De Biasio A / Rashid F / Merino N / Ragan TJ / Savva C / Zaher MS / Blanco FJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Structure of the processive human Pol δ holoenzyme.
Authors: Claudia Lancey / Muhammad Tehseen / Vlad-Stefan Raducanu / Fahad Rashid / Nekane Merino / Timothy J Ragan / Christos G Savva / Manal S Zaher / Afnan Shirbini / Francisco J Blanco / Samir M ...Authors: Claudia Lancey / Muhammad Tehseen / Vlad-Stefan Raducanu / Fahad Rashid / Nekane Merino / Timothy J Ragan / Christos G Savva / Manal S Zaher / Afnan Shirbini / Francisco J Blanco / Samir M Hamdan / Alfredo De Biasio /
Abstract: In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki ...In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ's activity in replicating the human genome.
History
DepositionJun 19, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseDec 25, 2019-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s1o
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10082.png

Downloads & links

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Map

FileDownload / File: emd_10082.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 Å		1.08 Å/pix.
x 360 pix.
= 388.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.015035675 - 0.03733129
Average (Standard dev.)0.00008226686 (±0.0011033687)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 388.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z388.800388.800388.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0150.0370.000

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Supplemental data

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Sample components

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Entire : PolD holoenzyme

EntireName: PolD holoenzyme
Components
  • Complex: PolD holoenzyme
    • Complex: Human polymerase delta
      • Protein or peptide: DNA polymerase delta catalytic subunit
      • Protein or peptide: DNA polymerase delta subunit 2
      • Protein or peptide: DNA polymerase delta subunit 3
      • Protein or peptide: DNA polymerase delta subunit 4
    • Complex: Proliferating cell nuclear antigen
      • Protein or peptide: Proliferating cell nuclear antigen
    • Complex: DNA
      • DNA: DNA primer
      • DNA: DNA template
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: PolD holoenzyme

SupramoleculeName: PolD holoenzyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5, #7

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Supramolecule #2: Human polymerase delta

SupramoleculeName: Human polymerase delta / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Proliferating cell nuclear antigen

SupramoleculeName: Proliferating cell nuclear antigen / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #6-#7
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: DNA polymerase delta catalytic subunit

MacromoleculeName: DNA polymerase delta catalytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 123.785922 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDGKRRPGPG PGVPPKRARG GLWDDDDAPR PSQFEEDLAL MEEMEAEHRL QEQEEEELQS VLEGVADGQV PPSAIDPRWL RPTPPALDP QTEPLIFQQL EIDHYVGPAQ PVPGGPPPSR GSVPVLRAFG VTDEGFSVCC HIHGFAPYFY TPAPPGFGPE H MGDLQREL ...String:
MDGKRRPGPG PGVPPKRARG GLWDDDDAPR PSQFEEDLAL MEEMEAEHRL QEQEEEELQS VLEGVADGQV PPSAIDPRWL RPTPPALDP QTEPLIFQQL EIDHYVGPAQ PVPGGPPPSR GSVPVLRAFG VTDEGFSVCC HIHGFAPYFY TPAPPGFGPE H MGDLQREL NLAISRDSRG GRELTGPAVL AVELCSRESM FGYHGHGPSP FLRITVALPR LVAPARRLLE QGIRVAGLGT PS FAPYEAN VDFEIRFMVD TDIVGCNWLE LPAGKYALRL KEKATQCQLE ADVLWSDVVS HPPEGPWQRI APLRVLSFDI ECA GRKGIF PEPERDPVIQ ICSLGLRWGE PEPFLRLALT LRPCAPILGA KVQSYEKEED LLQAWSTFIR IMDPDVITGY NIQN FDLPY LISRAQTLKV QTFPFLGRVA GLCSNIRDSS FQSKQTGRRD TKVVSMVGRV QMDMLQVLLR EYKLRSYTLN AVSFH FLGE QKEDVQHSII TDLQNGNDQT RRRLAVYCLK DAYLPLRLLE RLMVLVNAVE MARVTGVPLS YLLSRGQQVK VVSQLL RQA MHEGLLMPVV KSEGGEDYTG ATVIEPLKGY YDVPIATLDF SSLYPSIMMA HNLCYTTLLR PGTAQKLGLT EDQFIRT PT GDEFVKTSVR KGLLPQILEN LLSARKRAKA ELAKETDPLR RQVLDGRQLA LKVSANSVYG FTGAQVGKLP CLEISQSV T GFGRQMIEKT KQLVESKYTV ENGYSTSAKV VYGDTDSVMC RFGVSSVAEA MALGREAADW VSGHFPSPIR LEFEKVYFP YLLISKKRYA GLLFSSRPDA HDRMDCKGLE AVRRDNCPLV ANLVTASLRR LLIDRDPEGA VAHAQDVISD LLCNRIDISQ LVITKELTR AASDYAGKQA HVELAERMRK RDPGSAPSLG DRVPYVIISA AKGVAAYMKS EDPLFVLEHS LPIDTQYYLE Q QLAKPLLR IFEPILGEGR AEAVLLRGDH TRCKTVLTGK VGGLLAFAKR RNCCIGCRTV LSHQGAVCEF CQPRESELYQ KE VSHLNAL EERFSRLWTQ CQRCQGSLHE DVICTSRDCP IFYMRKKVRK DLEDQEQLLR RFGPPGPEAW

UniProtKB: DNA polymerase delta catalytic subunit

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Macromolecule #2: DNA polymerase delta subunit 2

MacromoleculeName: DNA polymerase delta subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.338168 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MFSEQAAQRA HTLLSPPSAN NATFARVPVA TYTNSSQPFR LGERSFSRQY AHIYATRLIQ MRPFLENRAQ QHWGSGVGVK KLCELQPEE KCCVVGTLFK AMPLQPSILR EVSEEHNLLP QPPRSKYIHP DDELVLEDEL QRIKLKGTID VSKLVTGTVL A VFGSVRDD ...String:
MFSEQAAQRA HTLLSPPSAN NATFARVPVA TYTNSSQPFR LGERSFSRQY AHIYATRLIQ MRPFLENRAQ QHWGSGVGVK KLCELQPEE KCCVVGTLFK AMPLQPSILR EVSEEHNLLP QPPRSKYIHP DDELVLEDEL QRIKLKGTID VSKLVTGTVL A VFGSVRDD GKFLVEDYCF ADLAPQKPAP PLDTDRFVLL VSGLGLGGGG GESLLGTQLL VDVVTGQLGD EGEQCSAAHV SR VILAGNL LSHSTQSRDS INKAKYLTKK TQAASVEAVK MLDEILLQLS ASVPVDVMPG EFDPTNYTLP QQPLHPCMFP LAT AYSTLQ LVTNPYQATI DGVRFLGTSG QNVSDIFRYS SMEDHLEILE WTLRVRHISP TAPDTLGCYP FYKTDPFIFP ECPH VYFCG NTPSFGSKII RGPEDQTVLL VTVPDFSATQ TACLVNLRSL ACQPISFSGF GAEDDDLGGL GLGP

UniProtKB: DNA polymerase delta subunit 2

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Macromolecule #3: DNA polymerase delta subunit 3

MacromoleculeName: DNA polymerase delta subunit 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.5285 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKA DQLYLENIDE FVTDQNKIVT YKWLSYTLGV HVNQAKQMLY DYVERKRKEN SGAQLHVTYL VSGSLIQNGH SCHKVAVVR EDKLEAVKSK LAVTASIHVY SIQKAMLKDS GPLFNTDYDI LKSNLQNCSK FSAIQCAAAV PRAPAESSSS S KKFEQSHL ...String:
MWSHPQFEKA DQLYLENIDE FVTDQNKIVT YKWLSYTLGV HVNQAKQMLY DYVERKRKEN SGAQLHVTYL VSGSLIQNGH SCHKVAVVR EDKLEAVKSK LAVTASIHVY SIQKAMLKDS GPLFNTDYDI LKSNLQNCSK FSAIQCAAAV PRAPAESSSS S KKFEQSHL HMSSETQANN ELTTNGHGPP ASKQVSQQPK GIMGMFASKA AAKTQETNKE TKTEAKEVTN ASAAGNKAPG KG NMMSNFF GKAAMNKFKV NLDSEQAVKE EKIVEQPTVS VTEPKLATPA GLKKSSKKAE PVKVLQKEKK RGKRVALSDD ETK ETENMR KKRRRIKLPE SDSSEDEVFP DSPGAYEAES PSPPPPPSPP LEPVPKTEPE PPSVKSSSGE NKRKRKRVLK SKTY LDGEG CIVTEKVYES ESCTDSEEEL NMKTSSVHRP PAMTVKKEPR EERKGPKKGT AALGKANRQV SITGFFQRK

UniProtKB: DNA polymerase delta subunit 3

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Macromolecule #4: DNA polymerase delta subunit 4

MacromoleculeName: DNA polymerase delta subunit 4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.274323 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MHHHHHHSRA WRHPQFGGHH HHHHENLYFQ SGRKRLITDS YPVVKRREGP AGHSKGELAP ELGEEPQPRD EEEAELELLR QFDLAWQYG PCTGITRLQR WCRAKQMGLE PPPEVWQVLK THPGDPRFQC SLWHLYPL

UniProtKB: DNA polymerase delta subunit 4

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Macromolecule #5: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 29.088061 KDa
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

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #6: DNA primer

MacromoleculeName: DNA primer / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.665987 KDa
SequenceString:
(DA)(DG)(DC)(DT)(DA)(DT)(DG)(DA)(DC)(DC) (DA)(DT)(DG)(DA)(DT)(DT)(DA)(DC)(DG)(DA) (DA)(DT)(DT)(DG)(DOC)

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Macromolecule #7: DNA template

MacromoleculeName: DNA template / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.677539 KDa
SequenceString:
(DC)(DT)(DG)(DC)(DA)(DC)(DG)(DA)(DA)(DT) (DT)(DA)(DA)(DG)(DC)(DA)(DA)(DT)(DT)(DC) (DG)(DT)(DA)(DA)(DT)(DC)(DA)(DT)(DG) (DG)(DT)(DC)(DA)(DT)(DA)(DG)(DC)(DT)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 9 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #10: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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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

Concentration0.355 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
100.0 mMCH3CO2KPotassium acetate
10.0 mMCaCl2Calcium chloride
0.02 %H(C2H4O)nO(C6H4)C9H19NP-40
0.4 mMC10H16N2O3SBiotin
1.0 mMC4H10O2S2Dithiothreitol
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec.
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 opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 3575 / Average exposure time: 60.0 sec. / Average electron dose: 35.0 e/Å2 / Details: 75 fractions per image
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.6 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 75000
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 initial model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8571
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 5
FSC plot (resolution estimation)

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