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Yorodumi- PDB-8unj: Atomic model of the human CTF18-RFC alone in the apo state (State 1) -
+Open data
-Basic information
Entry | Database: PDB / ID: 8unj | |||||||||
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Title | Atomic model of the human CTF18-RFC alone in the apo state (State 1) | |||||||||
Components |
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Keywords | REPLICATION / DNA clamp loader complex | |||||||||
Function / homology | Function and homology information response to organophosphorus / Ctf18 RFC-like complex / Rad17 RFC-like complex / Elg1 RFC-like complex / DNA replication factor C complex / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / DNA clamp loader activity / Polymerase switching on the C-strand of the telomere / single-stranded DNA helicase activity ...response to organophosphorus / Ctf18 RFC-like complex / Rad17 RFC-like complex / Elg1 RFC-like complex / DNA replication factor C complex / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / DNA clamp loader activity / Polymerase switching on the C-strand of the telomere / single-stranded DNA helicase activity / 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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.35 Å | |||||||||
Authors | Wang, F. / He, Q. / Li, H. | |||||||||
Funding support | United States, 2items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8unj.cif.gz | 291.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8unj.ent.gz | 221.6 KB | Display | PDB format |
PDBx/mmJSON format | 8unj.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/un/8unj ftp://data.pdbj.org/pub/pdb/validation_reports/un/8unj | HTTPS FTP |
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-Related structure data
Related structure data | 42406MC 8umtC 8umuC 8umvC 8umwC 8umyC 8un0C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 1 types, 1 molecules A
#1: Protein | Mass: 107523.984 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CHTF18 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: Q8WVB6 |
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-Replication factor C subunit ... , 4 types, 4 molecules BCDE
#2: Protein | Mass: 39203.207 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC2 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P35250 |
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#3: Protein | Mass: 38545.512 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC5 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P40937 |
#4: Protein | Mass: 39735.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC4 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P35249 |
#5: Protein | Mass: 40614.332 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC3 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P40938 |
-Non-polymers , 3 types, 7 molecules
#6: Chemical | #7: Chemical | #8: Chemical | ChemComp-ADP / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: the human clamp loader CTF18_RFC / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT |
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Molecular weight | Value: 0.3 MDa / Experimental value: YES |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Insect cell expression vector pTIE1 (others) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1900 nm / Nominal defocus min: 1200 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90327 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 123 / Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||
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