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- EMDB-41665: Cryo-EM structure of S. cerevisiae Ctf18-RFC-PCNA-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-41665
TitleCryo-EM structure of S. cerevisiae Ctf18-RFC-PCNA-DNA complex
Map data
Sample
  • Complex: Ctf18-RFC-PCNA complex
    • Protein or peptide: x 6 types
    • DNA: x 2 types
  • Ligand: x 3 types
KeywordsCtf18 / RFC2-5 / PCNA / DNA / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


positive regulation of DNA metabolic process / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Elg1 RFC-like complex / Removal of the Flap Intermediate / DNA replication factor C complex / telomere tethering at nuclear periphery ...positive regulation of DNA metabolic process / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Elg1 RFC-like complex / Removal of the Flap Intermediate / DNA replication factor C complex / telomere tethering at nuclear periphery / Ctf18 RFC-like complex / Polymerase switching / E3 ubiquitin ligases ubiquitinate target proteins / maintenance of DNA trinucleotide repeats / SUMOylation of DNA replication proteins / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Translesion Synthesis by POLH / DNA replication checkpoint signaling / establishment of mitotic sister chromatid cohesion / Termination of translesion DNA synthesis / Activation of ATR in response to replication stress / PCNA complex / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / error-free translesion synthesis / DNA polymerase processivity factor activity / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / nuclear replication fork / Dual incision in TC-NER / DNA replication initiation / translesion synthesis / subtelomeric heterochromatin formation / mismatch repair / positive regulation of DNA repair / DNA damage checkpoint signaling / positive regulation of DNA replication / replication fork / double-strand break repair via homologous recombination / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / DNA repair / ATP hydrolysis activity / mitochondrion / DNA binding / ATP binding / identical protein binding / nucleus / cytosol
Similarity search - Function
RFC1-like, AAA+ ATPase lid domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. ...RFC1-like, AAA+ ATPase lid domain / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / 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 / : / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Proliferating cell nuclear antigen / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2 / Chromosome transmission fidelity protein 18
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYuan Z / Georgescu R / O'Donnell M / Li H
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115809 United States
Howard Hughes Medical Institute (HHMI)M.E.O. United States
CitationJournal: Science / Year: 2024
Title: Mechanism of PCNA loading by Ctf18-RFC for leading-strand DNA synthesis.
Authors: Zuanning Yuan / Roxana Georgescu / Nina Y Yao / Olga Yurieva / Michael E O'Donnell / Huilin Li /
Abstract: The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific ...The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Polε). We reveal here the underlying mechanism of Ctf18-RFC specificity to Polε using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Polε contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Polε to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Polε off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Polε. These elements in both Ctf18-RFC and Polε provide specificity in loading PCNA onto DNA for Polε.
History
DepositionAug 20, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_41665.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.9472442 - 1.7732081
Average (Standard dev.)0.0091451155 (±0.06701236)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.968 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ctf18-RFC-PCNA complex

EntireName: Ctf18-RFC-PCNA complex
Components
  • Complex: Ctf18-RFC-PCNA complex
    • Protein or peptide: Replication factor C subunit 4
    • Protein or peptide: Replication factor C subunit 3
    • Protein or peptide: Replication factor C subunit 2
    • Protein or peptide: Replication factor C subunit 5
    • Protein or peptide: Chromosome transmission fidelity protein 18
    • Protein or peptide: Proliferating cell nuclear antigen
    • DNA: Template DNA
    • DNA: Primer DNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Ctf18-RFC-PCNA complex

SupramoleculeName: Ctf18-RFC-PCNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Replication factor C subunit 4

MacromoleculeName: Replication factor C subunit 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 35.782516 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: TLSLQLPWVE KYRPQVLSDI VGNKETIDRL QQIAKDGNMP HMIISGMPGI GKTTSVHCLA HELLGRSYAD GVLELNASDD RGIDVVRNQ IKHFAQKKLH LPPGKHKIVI LDEADSMTAG AQQALRRTME LYSNSTRFAF ACNQSNKIIE PLQSRCAILR Y SKLSDEDV ...String:
TLSLQLPWVE KYRPQVLSDI VGNKETIDRL QQIAKDGNMP HMIISGMPGI GKTTSVHCLA HELLGRSYAD GVLELNASDD RGIDVVRNQ IKHFAQKKLH LPPGKHKIVI LDEADSMTAG AQQALRRTME LYSNSTRFAF ACNQSNKIIE PLQSRCAILR Y SKLSDEDV LKRLLQIIKL EDVKYTNDGL EAIIFTAEGD MRQAINNLQS TVAGHGLVNA DNVFKIVDSP HPLIVKKMLL AS NLEDSIQ ILRTDLWKKG YSSIDIVTTS FRVTKNLAQV KESVRLEMIK EIGLTHMRIL EGVGTYLQLA SMLAKIHKLN NK

UniProtKB: Replication factor C subunit 4

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Macromolecule #2: Replication factor C subunit 3

MacromoleculeName: Replication factor C subunit 3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 36.818879 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: SKENLPWVEK YRPETLDEVY GQNEVITTVR KFVDEGKLPH LLFYGPPGTG KTSTIVALAR EIYGKNYSNM VLELNASDDR GIDVVRNQI KDFASTRQIF SKGFKLIILD EADAMTNAAQ NALRRVIERY TKNTRFCVLA NYAHKLTPAL LSRCTRFRFQ P LPQEAIER ...String:
SKENLPWVEK YRPETLDEVY GQNEVITTVR KFVDEGKLPH LLFYGPPGTG KTSTIVALAR EIYGKNYSNM VLELNASDDR GIDVVRNQI KDFASTRQIF SKGFKLIILD EADAMTNAAQ NALRRVIERY TKNTRFCVLA NYAHKLTPAL LSRCTRFRFQ P LPQEAIER RIANVLVHEK LKLSPNAEKA LIELSNGDMR RVLNVLQSCK ATLDNPDEDE ISDDVIYECC GAPRPSDLKA VL KSILEDD WGTAHYTLNK VRSAKGLALI DLIEGIVKIL EDYELQNEET RVHLLTKLAD IEYSISKGGN DQIQGSAVIG AIK ASFENE T

UniProtKB: Replication factor C subunit 3

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Macromolecule #3: Replication factor C subunit 2

MacromoleculeName: Replication factor C subunit 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 38.256617 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: SKLAAEQSLA QQPWVEKYRP KNLDEVTAQD HAVTVLKKTL KSANLPHMLF YGPPGTGKTS TILALTKELY GPDLMKSRIL ELNASDERG ISIVREKVKN FARLTVSKPS KHDLENYPCP PYKIIILDEA DSMTADAQSA LRRTMETYSG VTRFCLICNY V TRIIDPLA ...String:
SKLAAEQSLA QQPWVEKYRP KNLDEVTAQD HAVTVLKKTL KSANLPHMLF YGPPGTGKTS TILALTKELY GPDLMKSRIL ELNASDERG ISIVREKVKN FARLTVSKPS KHDLENYPCP PYKIIILDEA DSMTADAQSA LRRTMETYSG VTRFCLICNY V TRIIDPLA SRCSKFRFKA LDASNAIDRL RFISEQENVK CDDGVLERIL DISAGDLRRG ITLLQSASKG AQYLGDGKNI TS TQVEELA GVVPHDILIE IVEKVKSGDF DEIKKYVNTF MKSGWSAASV VNQLHEYYIT NDNFDTNFKN QISWLLFTTD SRL NNGTNE HIQLLNLLVK ISQL

UniProtKB: Replication factor C subunit 2

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Macromolecule #4: Replication factor C subunit 5

MacromoleculeName: Replication factor C subunit 5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.993582 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVS SPYHLEITPS DMGNNDRIVI QELLKEVAQM EQVDFQDSKD GLAHRYKCVI INEANSLTKD AQAALRRTME K YSKNIRLI ...String:
MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVS SPYHLEITPS DMGNNDRIVI QELLKEVAQM EQVDFQDSKD GLAHRYKCVI INEANSLTKD AQAALRRTME K YSKNIRLI MVCDSMSPII APIKSRCLLI RCPAPSDSEI STILSDVVTN ERIQLETKDI LKRIAQASNG NLRVSLLMLE SM ALNNELA LKSSSPIIKP DWIIVIHKLT RKIVKERSVN SLIECRAVLY DLLAHCIPAN IILKELTFSL LDVETLNTTN KSS IIEYSS VFDERLSLGN KAIFHLEGFI AKVMCCLD

UniProtKB: Replication factor C subunit 5

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Macromolecule #5: Chromosome transmission fidelity protein 18

MacromoleculeName: Chromosome transmission fidelity protein 18 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 30.1825 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: NTWASSNKDS PISWFKIVNQ LFRKDPHRDI KEQFYELLNQ VELNGNSDRI LQGCFNIFPY VKYSDNGIRK PANISDWLFF HDLMYQSMY AHNGELLRYS ALVPLVFFQT FGDIANKDDI RMKNSEYEQR ELKRANSDIV SLIMRHISVQ SPLMASFTDR K SLIFEILP ...String:
NTWASSNKDS PISWFKIVNQ LFRKDPHRDI KEQFYELLNQ VELNGNSDRI LQGCFNIFPY VKYSDNGIRK PANISDWLFF HDLMYQSMY AHNGELLRYS ALVPLVFFQT FGDIANKDDI RMKNSEYEQR ELKRANSDIV SLIMRHISVQ SPLMASFTDR K SLIFEILP YLDSMISSDF NKIRNLKLKQ AIMEELVQLL KSFQLNLIQN RSEGFDVRGG LTIDPPIDEV VLLNPKHINE VQ HKRANNL SSLLAKIEEN R

UniProtKB: Chromosome transmission fidelity protein 18

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Macromolecule #6: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 28.944051 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MLEAKFEEAS LFKRIIDGFK DCVQLVNFQC KEDGIIAQAV DDSRVLLVSL EIGVEAFQEY RCDHPVTLGM DLTSLSKILR CGNNTDTLT LIADNTPDSI ILLFEDTKKD RIAEYSLKLM DIDADFLKIE ELQYDSTLSL PSSEFSKIVR DLSQLSDSIN I MITKETIK ...String:
MLEAKFEEAS LFKRIIDGFK DCVQLVNFQC KEDGIIAQAV DDSRVLLVSL EIGVEAFQEY RCDHPVTLGM DLTSLSKILR CGNNTDTLT LIADNTPDSI ILLFEDTKKD RIAEYSLKLM DIDADFLKIE ELQYDSTLSL PSSEFSKIVR DLSQLSDSIN I MITKETIK FVADGDIGSG SVIIKPFVDM EHPETSIKLE MDQPVDLTFG AKYLLDIIKG SSLSDRVGIR LSSEAPALFQ FD LKSGFLQ FFLAPKFNDE E

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #7: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.542633 KDa
SequenceString:
(DA)(DG)(DC)(DA)(DG)(DC)(DA)(DA)(DC)(DA) (DG)(DA)(DG)(DA)(DA)(DT)(DC)(DT)

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Macromolecule #8: Primer DNA

MacromoleculeName: Primer DNA / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.184356 KDa
SequenceString:
(DA)(DG)(DA)(DT)(DT)(DC)(DT)(DC)(DT)(DG) (DT)(DT)(DG)(DC)(DT)(DG)(DC)

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Macromolecule #9: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 9 / Number of copies: 3 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #11: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 11 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 115411
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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