+Open data
-Basic information
Entry | Database: PDB / ID: 8dr7 | ||||||||||||
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Title | Open state of RFC:PCNA bound to a nicked dsDNA | ||||||||||||
Components |
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Keywords | REPLICATION/DNA / REPLICATION-DNA complex | ||||||||||||
Function / homology | Function and homology information DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 ...DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Rad17 RFC-like complex / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / PCNA complex / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / sister chromatid cohesion / mitotic sister chromatid cohesion / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / mismatch repair / translesion synthesis / 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 | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||||||||
Authors | Schrecker, M. / Hite, R.K. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Elife / Year: 2022 Title: Multistep loading of a DNA sliding clamp onto DNA by replication factor C. Authors: Marina Schrecker / Juan C Castaneda / Sujan Devbhandari / Charanya Kumar / Dirk Remus / Richard K Hite / Abstract: The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader ...The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8dr7.cif.gz | 1023.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8dr7.ent.gz | 842.8 KB | Display | PDB format |
PDBx/mmJSON format | 8dr7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8dr7_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 8dr7_full_validation.pdf.gz | 1.8 MB | Display | |
Data in XML | 8dr7_validation.xml.gz | 84.6 KB | Display | |
Data in CIF | 8dr7_validation.cif.gz | 124.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/dr/8dr7 ftp://data.pdbj.org/pub/pdb/validation_reports/dr/8dr7 | HTTPS FTP |
-Related structure data
Related structure data | 27673MC 8dqwC 8dqxC 8dqzC 8dr0C 8dr1C 8dr3C 8dr4C 8dr5C 8dr6C 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
-Replication factor C subunit ... , 5 types, 5 molecules ABCDE
#1: Protein | Mass: 101689.086 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: RFC1, CDC44, YOR217W, YOR50-7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38630 |
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#2: Protein | Mass: 36201.039 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: RFC4, YOL094C, O0923 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339 |
#3: Protein | Mass: 38254.543 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: RFC3, YNL290W, N0533 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629 |
#4: Protein | Mass: 39794.473 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: RFC2, YJR068W, J1808 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348 |
#5: Protein | Mass: 39993.582 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: RFC5, YBR087W, YBR0810 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251 |
-Protein , 1 types, 3 molecules FGH
#6: Protein | Mass: 30991.285 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: PCNA, POL30, GI527_G0000300 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6B7JGY6 |
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-DNA chain , 3 types, 3 molecules IJK
#7: DNA chain | Mass: 7844.004 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) |
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#8: DNA chain | Mass: 3585.320 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) |
#9: DNA chain | Mass: 3560.307 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) |
-Non-polymers , 3 types, 9 molecules
#10: Chemical | ChemComp-AGS / #11: Chemical | ChemComp-MG / #12: Chemical | ChemComp-GDP / | |
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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: Open state of RFC:PCNA bound to a nicked dsDNA / Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT | ||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||
Buffer solution | pH: 7.6 | ||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||
Specimen support | Grid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm |
Image recording | Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20.1_4487: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142294 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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