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Yorodumi- PDB-7tic: Structure of the yeast clamp loader (Replication Factor C RFC) bo... -
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-Basic information
Entry | Database: PDB / ID: 7tic | |||||||||||||||
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Title | Structure of the yeast clamp loader (Replication Factor C RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen PCNA) in an autoinhibited conformation | |||||||||||||||
Components |
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Keywords | REPLICATION / sliding clamp / DNA replication / AAA+ / clamp loader | |||||||||||||||
Function / homology | Function and homology information DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Rad17 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex ...DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Rad17 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching / SUMOylation of DNA replication proteins / positive regulation of DNA metabolic process / maintenance of DNA trinucleotide repeats / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / PCNA complex / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / error-free translesion synthesis / DNA polymerase processivity factor activity / positive regulation of DNA replication / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / positive regulation of DNA repair / mismatch repair / translesion synthesis / DNA damage checkpoint signaling / replication fork / 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 | |||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||||||||
Authors | Gaubitz, C. / Liu, X. / Pajak, J. / Stone, N. / Hayes, J. / Demo, G. / Kelch, B.A. | |||||||||||||||
Funding support | United States, Switzerland, Czech Republic, 4items
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Citation | Journal: 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 |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7tic.cif.gz | 460.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7tic.ent.gz | 367.8 KB | Display | PDB format |
PDBx/mmJSON format | 7tic.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7tic_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 7tic_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 7tic_validation.xml.gz | 77.4 KB | Display | |
Data in CIF | 7tic_validation.cif.gz | 115.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ti/7tic ftp://data.pdbj.org/pub/pdb/validation_reports/ti/7tic | HTTPS FTP |
-Related structure data
Related structure data | 25569MC 7thjC 7thvC 7ti8C 7tibC 7tidC 7tkuC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-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: 95048.195 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: Escherichia coli BL21(DE3) (bacteria) / 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: Escherichia coli BL21(DE3) (bacteria) / 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: Escherichia coli BL21(DE3) (bacteria) / 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: Escherichia coli BL21(DE3) (bacteria) / 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: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P38251 |
-Protein , 1 types, 3 molecules FGH
#6: Protein | Mass: 29525.713 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast) Gene: POL30, YBR088C, YBR0811 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15873 |
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-Non-polymers , 3 types, 9 molecules
#7: Chemical | ChemComp-AGS / #8: Chemical | ChemComp-MG / #9: 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: Replication Factor C, eukaryotic clamp loader / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT |
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Molecular weight | Value: 0.336 MDa / Experimental value: NO |
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1100 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 6109 |
-Processing
Software | Name: PHENIX / Version: dev_3699: / 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: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68227 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 1SXJ Accession code: 1SXJ / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
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