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- EMDB-42406: Cryo-EM map of the human CTF18-RFC alone in the apo state (State 1) -

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Basic information

Entry
Database: EMDB / ID: EMD-42406
TitleCryo-EM map of the human CTF18-RFC alone in the apo state (State 1)
Map dataPrimary sharpen EM map
Sample
  • Complex: the human clamp loader CTF18_RFC
    • Protein or peptide: Chromosome transmission fidelity protein 18 homolog
    • Protein or peptide: Replication factor C subunit 2
    • Protein or peptide: Replication factor C subunit 5
    • Protein or peptide: Replication factor C subunit 4
    • Protein or peptide: Replication factor C subunit 3
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsDNA clamp loader complex / REPLICATION
Function / homology
Function and homology information


Elg1 RFC-like complex / response to organophosphorus / DNA replication factor C complex / Ctf18 RFC-like complex / DNA clamp loader activity / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Polymerase switching on the C-strand of the telomere / DNA strand elongation involved in DNA replication / DNA duplex unwinding ...Elg1 RFC-like complex / response to organophosphorus / DNA replication factor C complex / Ctf18 RFC-like complex / DNA clamp loader activity / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Polymerase switching on the C-strand of the telomere / DNA strand elongation involved in DNA replication / DNA duplex unwinding / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / DNA synthesis involved in DNA repair / Presynaptic phase of homologous DNA pairing and strand exchange / PCNA-Dependent Long Patch Base Excision Repair / ATP-dependent activity, acting on DNA / Activation of ATR in response to replication stress / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / G2/M DNA damage checkpoint / Dual Incision in GG-NER / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / Processing of DNA double-strand break ends / Regulation of TP53 Activity through Phosphorylation / DNA replication / cell cycle / DNA repair / enzyme binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / membrane / nucleus / cytosol
Similarity search - Function
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 / 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 4 / Replication factor C subunit 2 / Replication factor C subunit 5 / Replication factor C subunit 3 / Chromosome transmission fidelity protein 18 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsWang F / He Q / Li H
Funding support United States, 2 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
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.
Authors: Qing He / Feng Wang / Michael E O'Donnell / Huilin Li /
Abstract: The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded ...The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique β-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.
History
DepositionOct 19, 2023-
Header (metadata) releaseMay 8, 2024-
Map releaseMay 8, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42406.png

Downloads & links

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Map

FileDownload / File: emd_42406.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary sharpen EM map
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-1.384913 - 2.0593402
Average (Standard dev.)-0.00062836334 (±0.04125729)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 264.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Original unsharpen EM map

Fileemd_42406_additional_1.map
AnnotationOriginal unsharpen EM map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpen EM map by DeepEMhancer

Fileemd_42406_additional_2.map
AnnotationSharpen EM map by DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map A

Fileemd_42406_half_map_1.map
AnnotationEM half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map B

Fileemd_42406_half_map_2.map
AnnotationEM half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : the human clamp loader CTF18_RFC

EntireName: the human clamp loader CTF18_RFC
Components
  • Complex: the human clamp loader CTF18_RFC
    • Protein or peptide: Chromosome transmission fidelity protein 18 homolog
    • Protein or peptide: Replication factor C subunit 2
    • Protein or peptide: Replication factor C subunit 5
    • Protein or peptide: Replication factor C subunit 4
    • Protein or peptide: Replication factor C subunit 3
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: the human clamp loader CTF18_RFC

SupramoleculeName: the human clamp loader CTF18_RFC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Chromosome transmission fidelity protein 18 homolog

MacromoleculeName: Chromosome transmission fidelity protein 18 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 107.523984 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MEDYEQELCG VEDDFHNQFA AELEVLAELE GASTPSPSGV PLFTAGRPPR TFEEALARGD AASSPAPAAS VGSSQGGARK RQVDADLQP AGSLPHAPRI KRPRLQVVKR LNFRSEEMEE PPPPDSSPTD ITPPPSPEDL AELWGHGVSE AAADVGLTRA S PAARNPVL ...String:
MEDYEQELCG VEDDFHNQFA AELEVLAELE GASTPSPSGV PLFTAGRPPR TFEEALARGD AASSPAPAAS VGSSQGGARK RQVDADLQP AGSLPHAPRI KRPRLQVVKR LNFRSEEMEE PPPPDSSPTD ITPPPSPEDL AELWGHGVSE AAADVGLTRA S PAARNPVL RRPPILEDYV HVTSTEGVRA YLVLRADPMA PGVQGSLLHV PWRGGGQLDL LGVSLASLKK QVDGERRERL LQ EAQKLSD TLHSLRSGEE EAAQPLGAPE EEPTDGQDAS SHCLWVDEFA PRHYTELLSD DFTNRCLLKW LKLWDLVVFG HER PSRKPR PSVEPARVSK EATAPGKWKS HEQVLEEMLE AGLDPSQRPK QKVALLCGPP GLGKTTLAHV IARHAGYSVV EMNA SDDRS PEVFRTRIEA ATQMESVLGA GGKPNCLVID EIDGAPVAAI NVLLSILNRK GPQEVGPQGP AVPSGGGRRR RAEGG LLMR PIICICNDQF APSLRQLKQQ AFLLHFPPTL PSRLVQRLQE VSLRQGMRAD PGVLAALCEK TDNDIRACIN TLQFLY SRG QRELSVRDVQ ATRVGLKDQR RGLFSVWQEV FQLPRAQRRR VGQDPALPAD TLLLGDGDAG SLTSASQRFY RVLHAAA SA GEHEKVVQGL FDNFLRLRLR DSSLGAVCVA LDWLAFDDLL AGAAHHSQSF QLLRYPPFLP VAFHVLFASS HTPRITFP S SQQEAQNRMS QMRNLIQTLV SGIAPATRSR ATPQALLLDA LCLLLDILAP KLRPVSTQLY STREKQQLAS LVGTMLAYS LTYRQERTPD GQYIYRLEPN VEELCRFPEL PARKPLTYQT KQLIAREIEV EKMRRAEASA RVENSPQVDG SPPGLEGLLG GIGEKGVHR PAPRNHEQRL EHIMRRAARE EQPEKDFFGR VVVRSTAVPS AGDTAPEQDS VERRMGTAVG RSEVWFRFNE G VSNAVRRS LYIRDLL

UniProtKB: Chromosome transmission fidelity protein 18 homolog

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Macromolecule #2: Replication factor C subunit 2

MacromoleculeName: Replication factor C subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.203207 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MEVEAVCGGA GEVEAQDSDP APAFSKAPGS AGHYELPWVE KYRPVKLNEI VGNEDTVSRL EVFAREGNVP NIIIAGPPGT GKTTSILCL ARALLGPALK DAMLELNASN DRGIDVVRNK IKMFAQQKVT LPKGRHKIII LDEADSMTDG AQQALRRTME I YSKTTRFA ...String:
MEVEAVCGGA GEVEAQDSDP APAFSKAPGS AGHYELPWVE KYRPVKLNEI VGNEDTVSRL EVFAREGNVP NIIIAGPPGT GKTTSILCL ARALLGPALK DAMLELNASN DRGIDVVRNK IKMFAQQKVT LPKGRHKIII LDEADSMTDG AQQALRRTME I YSKTTRFA LACNASDKII EPIQSRCAVL RYTKLTDAQI LTRLMNVIEK ERVPYTDDGL EAIIFTAQGD MRQALNNLQS TF SGFGFIN SENVFKVCDE PHPLLVKEMI QHCVNANIDE AYKILAHLWH LGYSPEDIIG NIFRVCKTFQ MAEYLKLEFI KEI GYTHMK IAEGVNSLLQ MAGLLARLCQ KTMAPVAS

UniProtKB: Replication factor C subunit 2

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Macromolecule #3: Replication factor C subunit 5

MacromoleculeName: Replication factor C subunit 5 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 38.545512 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: METSALKQQE QPAATKIRNL PWVEKYRPQT LNDLISHQDI LSTIQKFINE DRLPHLLLYG PPGTGKTSTI LACAKQLYKD KEFGSMVLE LNASDDRGID IIRGPILSFA STRTIFKKGF KLVILDEADA MTQDAQNALR RVIEKFTENT RFCLICNYLS K IIPALQSR ...String:
METSALKQQE QPAATKIRNL PWVEKYRPQT LNDLISHQDI LSTIQKFINE DRLPHLLLYG PPGTGKTSTI LACAKQLYKD KEFGSMVLE LNASDDRGID IIRGPILSFA STRTIFKKGF KLVILDEADA MTQDAQNALR RVIEKFTENT RFCLICNYLS K IIPALQSR CTRFRFGPLT PELMVPRLEH VVEEEKVDIS EDGMKALVTL SSGDMRRALN ILQSTNMAFG KVTEETVYTC TG HPLKSDI ANILDWMLNQ DFTTAYRNIT ELKTLKGLAL HDILTEIHLF VHRVDFPSSV RIHLLTKMAD IEYRLSVGTN EKI QLSSLI AAFQVTRDLI VAEA

UniProtKB: Replication factor C subunit 5

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Macromolecule #4: Replication factor C subunit 4

MacromoleculeName: Replication factor C subunit 4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 39.735711 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MQAFLKGTSI STKPPLTKDR GVAASAGSSG ENKKAKPVPW VEKYRPKCVD EVAFQEEVVA VLKKSLEGAD LPNLLFYGPP GTGKTSTIL AAARELFGPE LFRLRVLELN ASDERGIQVV REKVKNFAQL TVSGSRSDGK PCPPFKIVIL DEADSMTSAA Q AALRRTME ...String:
MQAFLKGTSI STKPPLTKDR GVAASAGSSG ENKKAKPVPW VEKYRPKCVD EVAFQEEVVA VLKKSLEGAD LPNLLFYGPP GTGKTSTIL AAARELFGPE LFRLRVLELN ASDERGIQVV REKVKNFAQL TVSGSRSDGK PCPPFKIVIL DEADSMTSAA Q AALRRTME KESKTTRFCL ICNYVSRIIE PLTSRCSKFR FKPLSDKIQQ QRLLDIAKKE NVKISDEGIA YLVKVSEGDL RK AITFLQS ATRLTGGKEI TEKVITDIAG VIPAEKIDGV FAACQSGSFD KLEAVVKDLI DEGHAATQLV NQLHDVVVEN NLS DKQKSI ITEKLAEVDK CLADGADEHL QLISLCATVM QQLSQNC

UniProtKB: Replication factor C subunit 4

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Macromolecule #5: Replication factor C subunit 3

MacromoleculeName: Replication factor C subunit 3 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.614332 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MSLWVDKYRP CSLGRLDYHK EQAAQLRNLV QCGDFPHLLV YGPSGAGKKT RIMCILRELY GVGVEKLRIE HQTITTPSKK KIEISTIAS NYHLEVNPSD AGNSDRVVIQ EMLKTVAQSQ QLETNSQRDF KVVLLTEVDK LTKDAQHALR RTMEKYMSTC R LILCCNST ...String:
MSLWVDKYRP CSLGRLDYHK EQAAQLRNLV QCGDFPHLLV YGPSGAGKKT RIMCILRELY GVGVEKLRIE HQTITTPSKK KIEISTIAS NYHLEVNPSD AGNSDRVVIQ EMLKTVAQSQ QLETNSQRDF KVVLLTEVDK LTKDAQHALR RTMEKYMSTC R LILCCNST SKVIPPIRSR CLAVRVPAPS IEDICHVLST VCKKEGLNLP SQLAHRLAEK SCRNLRKALL MCEACRVQQY PF TADQEIP ETDWEVYLRE TANAIVSQQT PQRLLEVRGR LYELLTHCIP PEIIMKGLLS ELLHNCDGQL KGEVAQMAAY YEH RLQLGS KAIYHLEAFV AKFMALYKKF MEDGLEGMMF

UniProtKB: Replication factor C subunit 3

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 7 / 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 #8: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / 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

Concentration0.3 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 280 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: RANDOM ASSIGNMENT
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 90327
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 123
Output model

PDB-8unj:
Atomic model of the human CTF18-RFC alone in the apo state (State 1)

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