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- EMDB-25614: Structure of the yeast clamp loader (Replication Factor C RFC) bo... -

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Entry
Database: EMDB / ID: EMD-25614
TitleStructure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation
Map dataSharpened Map
Sample
  • Complex: Replication Factor C (RFC), the eukaryotic clamp loader
    • Protein or peptide: PCNAProliferating cell nuclear antigen
    • Protein or peptide: Rfc1
    • Protein or peptide: Rfc2
    • Protein or peptide: Rfc3
    • Protein or peptide: Rfc4
    • RNA: Rfc5
Keywordssliding clamp / DNA replication / AAA+ / clamp loader / REPLICATION
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA clamp loader activity ...DNA clamp unloading / Rad17 RFC-like complex / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / meiotic mismatch repair / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA clamp loader activity / Polymerase switching / SUMOylation of DNA replication proteins / positive regulation of DNA metabolic process / maintenance of DNA trinucleotide repeats / PCNA complex / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / leading strand elongation / DNA polymerase processivity factor activity / error-free translesion synthesis / Gap-filling DNA repair synthesis and ligation in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA repair / replication fork / positive regulation of DNA replication / DNA damage checkpoint signaling / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / identical protein 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 / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site ...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 / 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 / : / 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
Proliferating cell nuclear antigen / 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 speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsGaubitz C / Demo G / Stone NP / Hayes JA / Liu X / Pajak J / Kelch BA
Funding support United States, Switzerland, Czech Republic, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM127776 United States
Swiss National Science Foundation168972 Switzerland
Swiss National Science Foundation177859 Switzerland
Ministry of Education (MoE, Czech Republic)LL2008 Czech Republic
CitationJournal: Elife / Year: 2022
Title: Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
Authors: Christl Gaubitz / Xingchen Liu / Joshua Pajak / Nicholas P Stone / Janelle A Hayes / Gabriel Demo / Brian A Kelch /
Abstract: Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. ...Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.
History
DepositionDec 5, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.9
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  • Surface view with fitted model
  • Atomic models: PDB-7thv
  • Surface level: 0.9
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25614.png

Downloads & links

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Map

FileDownload / File: emd_25614.map.gz / Format: CCP4 / Size: 8.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened Map
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-8.168092 - 12.871487
Average (Standard dev.)-0.000000000015238 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin505663
Dimensions140127119
Spacing119140127
CellA: 126.021 Å / B: 148.26001 Å / C: 134.493 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z119140127
origin x/y/z0.0000.0000.000
length x/y/z126.021148.260134.493
α/β/γ90.00090.00090.000
start NX/NY/NZ635056
NX/NY/NZ119140127
MAP C/R/S321
start NC/NR/NS565063
NC/NR/NS127140119
D min/max/mean-8.16812.871-0.000

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Supplemental data

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Additional map: Unsharpened Map

Fileemd_25614_additional_1.map
AnnotationUnsharpened Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_25614_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25614_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 : Replication Factor C (RFC), the eukaryotic clamp loader

EntireName: Replication Factor C (RFC), the eukaryotic clamp loader
Components
  • Complex: Replication Factor C (RFC), the eukaryotic clamp loader
    • Protein or peptide: PCNAProliferating cell nuclear antigen
    • Protein or peptide: Rfc1
    • Protein or peptide: Rfc2
    • Protein or peptide: Rfc3
    • Protein or peptide: Rfc4
    • RNA: Rfc5

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Supramolecule #1: Replication Factor C (RFC), the eukaryotic clamp loader

SupramoleculeName: Replication Factor C (RFC), the eukaryotic clamp loader
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 336 KDa

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Macromolecule #1: PCNA

MacromoleculeName: PCNA / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GPHMASMLEA KFEEASLFKR IIDGFKDCV QLVNFQCKED G IIAQAVDD SRVLLVSLEI GV EAFQEYR CDHPVTLGMD LTS LSKILR CGNNTDTLTL IADN TPDSI ILLFEDTKKD RIAEY SLKL MDIDADFLKI EELQYD STL SLPSSEFSKI VRDLSQL SD ...String:
GPHMASMLEA KFEEASLFKR IIDGFKDCV QLVNFQCKED G IIAQAVDD SRVLLVSLEI GV EAFQEYR CDHPVTLGMD LTS LSKILR CGNNTDTLTL IADN TPDSI ILLFEDTKKD RIAEY SLKL MDIDADFLKI EELQYD STL SLPSSEFSKI VRDLSQL SD SINIMITKET IKFVADGD I GSGSVIIKPF VDMEHPETS IKLEMDQPVD LTFGAKYLLD IIKGSSLSD RVGIRLSSEA P ALFQFDLK SGFLQFFLAP KF NDEE

UniProtKB: Proliferating cell nuclear antigen

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Macromolecule #2: Rfc1

MacromoleculeName: Rfc1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MVNISDFFGK NKKSVRSSTS RPTRQVGSSK PEVIDLDTES DQESTNKTPK KMPVSNVIDV SETPEGEKKL PLPAKRKASS PTVKPASSKK TKPSSKSSDS ASNITAQDVL DKIPSLDLSN VHVKENAKFD FKSANSNADP DEIVSEIGSF PEGKPNCLLG LTIVFTGVLP ...String:
MVNISDFFGK NKKSVRSSTS RPTRQVGSSK PEVIDLDTES DQESTNKTPK KMPVSNVIDV SETPEGEKKL PLPAKRKASS PTVKPASSKK TKPSSKSSDS ASNITAQDVL DKIPSLDLSN VHVKENAKFD FKSANSNADP DEIVSEIGSF PEGKPNCLLG LTIVFTGVLP TLERGASEAL AKRYGARVTK SISSKTSVVV LGDEAGPKKL EKIKQLKIKA IDEEGFKQLI AGMPAEGGDG EAAEKARRKL EEQHNIATKE AELLVKKEEE RSKKLAATRV SGGHLERDNV VREEDKLWTV KYAPTNLQQV CGNKGSVMKL KNWLANWENS KKNSFKHAGK DGSGVFRAAM LYGPPGIGKT TAAHLVAQEL GYDILEQNAS DVRSKTLLNA GVKNALDNMS VVGYFKHNEE AQNLNGKHFV IIMDEVDGMS GGDRGGVGQL AQFCRKTSTP LILICNERNL PKMRPFDRVC LDIQFRRPDA NSIKSRLMTI AIREKFKLDP NVIDRLIQTT RGDIRQVINL LSTISTTTKT INHENINEIS KAWEKNIALK PFDIAHKMLD GQIYSDIGSR NFTLNDKIAL YFDDFDFTPL MIQENYLSTR PSVLKPGQSH LEAVAEAANC ISLGDIVEKK IRSSEQLWSL LPLHAVLSSV YPASKVAGHM AGRINFTAWL GQNSKSAKYY RLLQEIHYHT RLGTSTDKIG LRLDYLPTFR KRLLDPFLKQ GADAISSVIE VMDDYYLTKE DWDSIMEFFV GPDVTTAIIK KIPATVKSGF TRKYNSMTHP VAIYRTGSTI GGGGVGTSTS TPDFEDVVDA DDNPVPADDE ETQDSSTDLK KDKLIKQKAK PTKRKTATSK PGGSKKRKTK A

UniProtKB: Replication factor C subunit 1

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Macromolecule #3: Rfc2

MacromoleculeName: Rfc2 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMKS RILELNASDE RGISIVREKV KNFARLTVSK PSKHDLENYP CPPYKIIILD EADSMTADAQ SALRRTMETY SGVTRFCLIC ...String:
MFEGFGPNKK RKISKLAAEQ SLAQQPWVEK YRPKNLDEVT AQDHAVTVLK KTLKSANLPH MLFYGPPGTG KTSTILALTK ELYGPDLMKS RILELNASDE RGISIVREKV KNFARLTVSK PSKHDLENYP CPPYKIIILD EADSMTADAQ SALRRTMETY SGVTRFCLIC NYVTRIIDPL ASRCSKFRFK ALDASNAIDR LRFISEQENV KCDDGVLERI LDISAGDLRR GITLLQSASK GAQYLGDGKN ITSTQVEELA GVVPHDILIE IVEKVKSGDF DEIKKYVNTF MKSGWSAASV VNQLHEYYIT NDNFDTNFKN QISWLLFTTD SRLNNGTNEH IQLLNLLVKI SQL

UniProtKB: Replication factor C subunit 2

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Macromolecule #4: Rfc3

MacromoleculeName: Rfc3 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRGI DVVRNQIKDF ASTRQIFSKG FKLIILDEAD AMTNAAQNAL RRVIERYTKN TRFCVLANYA HKLTPALLSR CTRFRFQPLP ...String:
MSTSTEKRSK ENLPWVEKYR PETLDEVYGQ NEVITTVRKF VDEGKLPHLL FYGPPGTGKT STIVALAREI YGKNYSNMVL ELNASDDRGI DVVRNQIKDF ASTRQIFSKG FKLIILDEAD AMTNAAQNAL RRVIERYTKN TRFCVLANYA HKLTPALLSR CTRFRFQPLP QEAIERRIAN VLVHEKLKLS PNAEKALIEL SNGDMRRVLN VLQSCKATLD NPDEDEISDD VIYECCGAPR PSDLKAVLKS ILEDDWGTAH YTLNKVRSAK GLALIDLIEG IVKILEDYEL QNEETRVHLL TKLADIEYSI SKGGNDQIQG SAVIGAIKAS FENETVKANV

UniProtKB: Replication factor C subunit 3

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Macromolecule #5: Rfc4

MacromoleculeName: Rfc4 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVVR NQIKHFAQKK LHLPPGKHKI VILDEADSMT AGAQQALRRT MELYSNSTRF AFACNQSNKI IEPLQSRCAI LRYSKLSDED ...String:
MSKTLSLQLP WVEKYRPQVL SDIVGNKETI DRLQQIAKDG NMPHMIISGM PGIGKTTSVH CLAHELLGRS YADGVLELNA SDDRGIDVVR NQIKHFAQKK LHLPPGKHKI VILDEADSMT AGAQQALRRT MELYSNSTRF AFACNQSNKI IEPLQSRCAI LRYSKLSDED VLKRLLQIIK LEDVKYTNDG LEAIIFTAEG DMRQAINNLQ STVAGHGLVN ADNVFKIVDS PHPLIVKKML LASNLEDSIQ ILRTDLWKKG YSSIDIVTTS FRVTKNLAQV KESVRLEMIK EIGLTHMRIL EGVGTYLQLA SMLAKIHKLN NKA

UniProtKB: Replication factor C subunit 4

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Macromolecule #6: Rfc5

MacromoleculeName: Rfc5 / type: rna / ID: 6
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVSS PYHLEITPSD MGNNDRIVIQ ELLKEVAQME QVDFQDSKDG LAHRYKCVII NEANSLTKDA QAALRRTMEK YSKNIRLIMV ...String:
MSLWVDKYRP KSLNALSHNE ELTNFLKSLS DQPRDLPHLL LYGPNGTGKK TRCMALLESI FGPGVYRLKI DVRQFVTASN RKLELNVVSS PYHLEITPSD MGNNDRIVIQ ELLKEVAQME QVDFQDSKDG LAHRYKCVII NEANSLTKDA QAALRRTMEK YSKNIRLIMV CDSMSPIIAP IKSRCLLIRC PAPSDSEIST ILSDVVTNER IQLETKDILK RIAQASNGNL RVSLLMLESM ALNNELALKS SSPIIKPDWI IVIHKLTRKI VKERSVNSLI ECRAVLYDLL AHCIPANIIL KELTFSLLDV ETLNTTNKSS IIEYSSVFDE RLSLGNKAIF HLEGFIAKVM CCLD

UniProtKB: Replication factor C subunit 5

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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

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 4 / Number real images: 6109 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 60036
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1sxj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7thv:
Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation

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