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- EMDB-25876: Cryo-EM 3D map of S. cerevisiae clamp loader RFC bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-25876
TitleCryo-EM 3D map of S. cerevisiae clamp loader RFC bound to DNA
Map data3D cryoEM map of yeast RFC-DNA1 complex
Sample
  • Complex: RFC-PCNA-DNA1-DNA2
    • Complex: dsDNADNA
      • DNA: Template strandTranscription (biology)
      • DNA: Primer strand
    • Complex: ProteinsProtein
      • Protein or peptide: Replication factor C subunit 1
      • 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
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / DNA clamp loader activity / Polymerase switching / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / sister chromatid cohesion ...DNA clamp unloading / Rad17 RFC-like complex / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / DNA clamp loader activity / Polymerase switching / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / mismatch repair / DNA damage checkpoint signaling / DNA-templated DNA replication / mitotic cell cycle / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal 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 / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain ...Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal 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 / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / 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
Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 1 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciessynthetic construct (others) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsZheng F / Georgescu R / Yao YN / O'Donnell ME / 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) United States
CitationJournal: Elife / Year: 2022
Title: Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair.
Authors: Fengwei Zheng / Roxana Georgescu / Nina Y Yao / Huilin Li / Michael E O'Donnell /
Abstract: RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during ...RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis.
History
DepositionJan 6, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25876.png

Downloads & links

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Map

FileDownload / File: emd_25876.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D cryoEM map of yeast RFC-DNA1 complex
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.8181001 - 1.4874514
Average (Standard dev.)-0.0006930265 (±0.027165415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 331.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : RFC-PCNA-DNA1-DNA2

EntireName: RFC-PCNA-DNA1-DNA2
Components
  • Complex: RFC-PCNA-DNA1-DNA2
    • Complex: dsDNADNA
      • DNA: Template strandTranscription (biology)
      • DNA: Primer strand
    • Complex: ProteinsProtein
      • Protein or peptide: Replication factor C subunit 1
      • 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
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: RFC-PCNA-DNA1-DNA2

SupramoleculeName: RFC-PCNA-DNA1-DNA2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#7

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Supramolecule #2: dsDNA

SupramoleculeName: dsDNA / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #6-#7 / Details: synthetic DNA
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: Proteins

SupramoleculeName: Proteins / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #1-#5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Replication factor C subunit 1

MacromoleculeName: Replication factor C subunit 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 95.048195 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MVNISDFFGK NKKSVRSSTS RPTRQVGSSK PEVIDLDTES DQESTNKTPK KMPVSNVIDV SETPEGEKKL PLPAKRKASS PTVKPASSK KTKPSSKSSD SASNITAQDV LDKIPSLDLS NVHVKENAKF DFKSANSNAD PDEIVSEIGS FPEGKPNCLL G LTIVFTGV ...String:
MVNISDFFGK NKKSVRSSTS RPTRQVGSSK PEVIDLDTES DQESTNKTPK KMPVSNVIDV SETPEGEKKL PLPAKRKASS PTVKPASSK KTKPSSKSSD SASNITAQDV LDKIPSLDLS NVHVKENAKF DFKSANSNAD PDEIVSEIGS FPEGKPNCLL G LTIVFTGV LPTLERGASE ALAKRYGARV TKSISSKTSV VVLGDEAGPK KLEKIKQLKI KAIDEEGFKQ LIAGMPAEGG DG EAAEKAR RKLEEQHNIA TKEAELLVKK EEERSKKLAA TRVSGGHLER DNVVREEDKL WTVKYAPTNL QQVCGNKGSV MKL KNWLAN WENSKKNSFK HAGKDGSGVF RAAMLYGPPG IGKTTAAHLV AQELGYDILE QNASDVRSKT LLNAGVKNAL DNMS VVGYF KHNEEAQNLN GKHFVIIMDE VDGMSGGDRG GVGQLAQFCR KTSTPLILIC NERNLPKMRP FDRVCLDIQF RRPDA NSIK SRLMTIAIRE KFKLDPNVID RLIQTTRGDI RQVINLLSTI STTTKTINHE NINEISKAWE KNIALKPFDI AHKMLD GQI YSDIGSRNFT LNDKIALYFD DFDFTPLMIQ ENYLSTRPSV LKPGQSHLEA VAEAANCISL GDIVEKKIRS SEQLWSL LP LHAVLSSVYP ASKVAGHMAG RINFTAWLGQ NSKSAKYYRL LQEIHYHTRL GTSTDKIGLR LDYLPTFRKR LLDPFLKQ G ADAISSVIEV MDDYYLTKED WDSIMEFFVG PDVTTAIIKK IPATVKSGFT RKYNSMTHPV AIYRTGSTIG GGGVGTSTS TPDFEDVVDA DDNPVPADDE ETQDSSTDLK KDKLIKQKAK PTKRKTATSK PGGSKKRKTK A

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Macromolecule #2: Replication factor C subunit 4

MacromoleculeName: Replication factor C subunit 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 36.201039 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVV RNQIKHFAQK KLHLPPGKHK IVILDEADSM TAGAQQALRR TMELYSNSTR FAFACNQSNK IIEPLQSRCA I LRYSKLSD ...String:
MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVV RNQIKHFAQK KLHLPPGKHK IVILDEADSM TAGAQQALRR TMELYSNSTR FAFACNQSNK IIEPLQSRCA I LRYSKLSD EDVLKRLLQI IKLEDVKYTN DGLEAIIFTA EGDMRQAINN LQSTVAGHGL VNADNVFKIV DSPHPLIVKK ML LASNLED SIQILRTDLW KKGYSSIDIV TTSFRVTKNL AQVKESVRLE MIKEIGLTHM RILEGVGTYL QLASMLAKIH KLN NKA

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Macromolecule #3: Replication factor C subunit 3

MacromoleculeName: Replication factor C subunit 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 38.254543 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRG IDVVRNQIKD FASTRQIFSK GFKLIILDEA DAMTNAAQNA LRRVIERYTK NTRFCVLANY AHKLTPALLS R CTRFRFQP ...String:
MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRG IDVVRNQIKD FASTRQIFSK GFKLIILDEA DAMTNAAQNA LRRVIERYTK NTRFCVLANY AHKLTPALLS R CTRFRFQP LPQEAIERRI ANVLVHEKLK LSPNAEKALI ELSNGDMRRV LNVLQSCKAT LDNPDEDEIS DDVIYECCGA PR PSDLKAV LKSILEDDWG TAHYTLNKVR SAKGLALIDL IEGIVKILED YELQNEETRV HLLTKLADIE YSISKGGNDQ IQG SAVIGA IKASFENETV KANV

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Macromolecule #4: Replication factor C subunit 2

MacromoleculeName: Replication factor C subunit 2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.794473 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMK SRILELNASD ERGISIVREK VKNFARLTVS KPSKHDLENY PCPPYKIIIL DEADSMTADA QSALRRTMET Y SGVTRFCL ...String:
MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMK SRILELNASD ERGISIVREK VKNFARLTVS KPSKHDLENY PCPPYKIIIL DEADSMTADA QSALRRTMET Y SGVTRFCL ICNYVTRIID PLASRCSKFR FKALDASNAI DRLRFISEQE NVKCDDGVLE RILDISAGDL RRGITLLQSA SK GAQYLGD GKNITSTQVE ELAGVVPHDI LIEIVEKVKS GDFDEIKKYV NTFMKSGWSA ASVVNQLHEY YITNDNFDTN FKN QISWLL FTTDSRLNNG TNEHIQLLNL LVKISQL

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Macromolecule #5: Replication factor C subunit 5

MacromoleculeName: Replication factor C subunit 5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 39.993582 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

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Macromolecule #6: Template strand

MacromoleculeName: Template strand / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.214818 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DA)(DT)(DG)(DT)(DA)(DC)(DT)(DC)(DG) (DT)(DA)(DG)(DT)(DG)(DT)(DC)(DT)(DG) (DC)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)

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Macromolecule #7: Primer strand

MacromoleculeName: Primer strand / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.136008 KDa
SequenceString:
(DG)(DC)(DA)(DG)(DA)(DC)(DA)(DC)(DT)(DA) (DC)(DG)(DA)(DG)(DT)(DA)(DC)(DA)(DT)(DA)

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Macromolecule #8: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 8 / Number of copies: 4 / 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 #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 10 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

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

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN 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: 1.9000000000000001 µm / Nominal defocus min: 1.3 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 65.0 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:

1sxj

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 334876

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