[English] 日本語
![](img/lk-miru.gif)
- PDB-6vvo: Structure of the human clamp loader (Replication Factor C, RFC) b... -
+
Open data
-
Basic information
Entry | Database: PDB / ID: 6vvo | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Structure of the human clamp loader (Replication Factor C, RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen, PCNA) | ||||||||||||
![]() |
| ||||||||||||
![]() | DNA BINDING PROTEIN / REPLICATION / sliding clamp / DNA replication / AAA+ / ATPase / clamp loader | ||||||||||||
Function / homology | ![]() DNA clamp unloader activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / response to organophosphorus / Ctf18 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity ...DNA clamp unloader activity / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / response to organophosphorus / Ctf18 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity / purine-specific mismatch base pair DNA N-glycosylase activity / nuclear lamina / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / replisome / DNA duplex unwinding / response to L-glutamate / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / DNA strand elongation involved in DNA replication / histone acetyltransferase binding / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / G1/S-Specific Transcription / response to dexamethasone / leading strand elongation / replication fork processing / nuclear replication fork / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / telomere maintenance via telomerase / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / ATP-dependent activity, acting on DNA / mismatch repair / translesion synthesis / Activation of ATR in response to replication stress / cyclin-dependent protein kinase holoenzyme complex / response to cadmium ion / DNA polymerase binding / enzyme activator activity / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / base-excision repair, gap-filling / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / positive regulation of DNA replication / replication fork / male germ cell nucleus / nuclear estrogen receptor binding / liver regeneration / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / Processing of DNA double-strand break ends / double-stranded DNA binding / DNA-binding transcription factor binding / DNA replication / Regulation of TP53 Activity through Phosphorylation / sequence-specific DNA binding / damaged DNA binding / chromosome, telomeric region / nuclear body / protein domain specific binding / DNA repair / centrosome / chromatin binding / chromatin / protein-containing complex binding / positive regulation of DNA-templated transcription / enzyme binding / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity Similarity search - Function | ||||||||||||
Biological species | ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | ||||||||||||
![]() | Gaubitz, C. / Liu, X. / Stone, N.P. / Kelch, B.A. | ||||||||||||
Funding support | ![]() ![]()
| ||||||||||||
![]() | ![]() Title: Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch. Authors: Christl Gaubitz / Xingchen Liu / Joseph Magrino / Nicholas P Stone / Jacob Landeck / Mark Hedglin / Brian A Kelch / ![]() Abstract: DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to ...DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health. | ||||||||||||
History |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | Molecule: ![]() ![]() |
-
Downloads & links
-
Download
PDBx/mmCIF format | ![]() | 499.8 KB | Display | ![]() |
---|---|---|---|---|
PDB format | ![]() | 393 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
---|
-Related structure data
Related structure data | ![]() 21405MC M: map data used to model this data C: citing same article ( |
---|---|
Similar structure data |
-
Links
-
Assembly
Deposited unit | ![]()
|
---|---|
1 |
|
-
Components
-Replication factor C subunit ... , 5 types, 5 molecules ABCDE
#1: Protein | Mass: 66221.227 Da / Num. of mol.: 1 / Fragment: UNP residues 556-1148 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
---|---|
#2: Protein | Mass: 39203.207 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
#3: Protein | Mass: 38545.512 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
#4: Protein | Mass: 39751.668 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
#5: Protein | Mass: 40614.332 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
-Protein , 1 types, 3 molecules FGH
#6: Protein | Mass: 28795.752 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() |
---|
-Non-polymers , 3 types, 9 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/AGS.gif)
![](data/chem/img/ADP.gif)
![](data/chem/img/AGS.gif)
![](data/chem/img/ADP.gif)
#7: Chemical | ChemComp-MG / #8: Chemical | ChemComp-AGS / #9: Chemical | ChemComp-ADP / | |
---|
-Details
Has ligand of interest | Y |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-
Sample preparation
Component | Name: Human Replication factor C (RFC) complex bound to the sliding clamp Proliferating cell nuclear antigen (PCNA) Type: COMPLEX Details: hRFC construct with a truncation of the A subunit's N-terminal region (missing residues 1-555) Entity ID: #1-#6 / Source: RECOMBINANT |
---|---|
Molecular weight | Value: 0.31 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R0.6/1 |
Vitrification | Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K |
-
Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company | |||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM imaging | Accelerating voltage: 300 kV / Electron source:
| |||||||||||||||||||||
Image recording |
|
-
Processing
Software |
| ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| ||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 193934 Details: Multi-body refinement was used to generate 2 reconstructions that were combined Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL Details: Initial local fitting was peformed using UCSF Chimera, followed by manual model adjustment, then refinement in PHENIX. | ||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 63.28 Å2 | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
|