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- PDB-6vvo: Structure of the human clamp loader (Replication Factor C, RFC) b... -

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Entry
Database: PDB / ID: 6vvo
TitleStructure of the human clamp loader (Replication Factor C, RFC) bound to the sliding clamp (Proliferating Cell Nuclear Antigen, PCNA)
Components
  • (Replication factor C subunit ...) x 5
  • Proliferating cell nuclear antigen
KeywordsDNA BINDING PROTEIN / REPLICATION / sliding clamp / DNA replication / AAA+ / ATPase / clamp loader
Function / homology
Function and homology information


DNA clamp unloader activity / response to organophosphorus / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity ...DNA clamp unloader activity / response to organophosphorus / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / 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 / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Removal of the Flap Intermediate from the C-strand / Transcription of E2F targets under negative control by DREAM complex / replisome / : / HDR through Single Strand Annealing (SSA) / response to L-glutamate / Impaired BRCA2 binding to RAD51 / histone acetyltransferase binding / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / replication fork processing / response to dexamethasone / nuclear replication fork / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / ATP-dependent activity, acting on DNA / PCNA-Dependent Long Patch Base Excision Repair / telomere maintenance via telomerase / mismatch repair / Activation of ATR in response to replication stress / translesion synthesis / response to cadmium ion / cyclin-dependent protein kinase holoenzyme complex / estrous cycle / DNA polymerase binding / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / enzyme activator activity / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / replication fork / male germ cell nucleus / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / receptor tyrosine kinase binding / Dual Incision in GG-NER / cellular response to hydrogen peroxide / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to UV / cellular response to xenobiotic stimulus / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / heart development / Processing of DNA double-strand break ends / double-stranded DNA binding / DNA-binding transcription factor binding / Regulation of TP53 Activity through Phosphorylation / DNA clamp loader activity / sequence-specific DNA binding / damaged DNA binding / DNA replication / chromosome, telomeric region / nuclear body / protein domain specific binding / DNA repair / centrosome / chromatin binding / protein-containing complex binding / chromatin / positive regulation of DNA-templated transcription / enzyme binding / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity
Similarity search - Function
Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / Zinc Finger, Delta Prime; domain 3 - #10 / Zinc Finger, Delta Prime; domain 3 / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : ...Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal domain / Zinc Finger, Delta Prime; domain 3 - #10 / Zinc Finger, Delta Prime; domain 3 / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / Helicase, Ruva Protein; domain 3 - #60 / BRCA1 C Terminus (BRCT) domain / : / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Up-down Bundle / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Proliferating cell nuclear antigen / Replication factor C subunit 4 / Replication factor C subunit 2 / Replication factor C subunit 1 / Replication factor C subunit 5 / Replication factor C subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGaubitz, C. / Liu, X. / Stone, N.P. / Kelch, B.A.
Funding support United States, Switzerland, 3items
OrganizationGrant numberCountry
American Cancer Society440685 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM127776 United States
Swiss National Science Foundation177859 Switzerland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
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
DepositionFeb 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 25, 2020Group: Data collection / Refinement description / Category: em_3d_fitting / em_image_recording / em_imaging
Item: _em_3d_fitting.details / _em_3d_fitting.ref_protocol / _em_image_recording.imaging_id
Revision 1.2Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / struct_conn
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21405
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Replication factor C subunit 1
B: Replication factor C subunit 2
C: Replication factor C subunit 5
D: Replication factor C subunit 4
E: Replication factor C subunit 3
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
hetero molecules


Theoretical massNumber of molelcules
Total (without water)313,34117
Polymers310,7238
Non-polymers2,6179
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Asymmetric - C1
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area35630 Å2
ΔGint-178 kcal/mol
Surface area98930 Å2

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Components

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Replication factor C subunit ... , 5 types, 5 molecules ABCDE

#1: Protein Replication factor C subunit 1 / Activator 1 140 kDa subunit / A1 140 kDa subunit / Activator 1 large subunit / Activator 1 subunit ...Activator 1 140 kDa subunit / A1 140 kDa subunit / Activator 1 large subunit / Activator 1 subunit 1 / DNA-binding protein PO-GA / Replication factor C 140 kDa subunit / RFC140 / Replication factor C large subunit


Mass: 66221.227 Da / Num. of mol.: 1 / Fragment: UNP residues 556-1148
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC1, RFC140 / Production host: Escherichia coli (E. coli) / References: UniProt: P35251
#2: Protein Replication factor C subunit 2 / Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 ...Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 kDa subunit / RFC40


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: Escherichia coli (E. coli) / References: UniProt: P35250
#3: Protein Replication factor C subunit 5 / Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 ...Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 kDa subunit / RFC36


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: Escherichia coli (E. coli) / References: UniProt: P40937
#4: Protein Replication factor C subunit 4 / Activator 1 37 kDa subunit / A1 37 kDa subunit / Activator 1 subunit 4 / Replication factor C 37 ...Activator 1 37 kDa subunit / A1 37 kDa subunit / Activator 1 subunit 4 / Replication factor C 37 kDa subunit / RFC37


Mass: 39751.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RFC4 / Production host: Escherichia coli (E. coli) / References: UniProt: P35249
#5: Protein Replication factor C subunit 3 / Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 ...Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 kDa subunit / RFC38


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: Escherichia coli (E. coli) / References: UniProt: P40938

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Protein , 1 types, 3 molecules FGH

#6: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


Mass: 28795.752 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA / Production host: Escherichia coli (E. coli) / References: UniProt: P12004

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Non-polymers , 3 types, 9 molecules

#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#9: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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 weightValue: 0.31 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R0.6/1
VitrificationCryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
EM imaging

Accelerating voltage: 300 kV / Electron source: FIELD EMISSION GUN / Illumination mode: FLOOD BEAM / Model: FEI TITAN KRIOS / Mode: BRIGHT FIELD / Cs: 2.7 mm / Specimen-ID: 1

ID
1
2
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelNum. of grids imagedNum. of real imagesDetails
1140.3GATAN K3 (6k x 4k)13695Quantifoil R2/2
2245GATAN K3 (6k x 4k)17840Quantifoil R0.6/1

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_3699refinement
PHENIXdev_3699refinement
EM software
IDNameVersionCategory
1cisTEM1.0.0particle selection
2SerialEMimage acquisition
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cisTEMinitial Euler assignment
11RELION3.0.2final Euler assignment
12RELION3.0.2classification
13RELION3.0.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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 buildingProtocol: OTHER / Space: REAL
Details: Initial local fitting was peformed using UCSF Chimera, followed by manual model adjustment, then refinement in PHENIX.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 63.28 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008319983
ELECTRON MICROSCOPYf_angle_d0.813127019
ELECTRON MICROSCOPYf_chiral_restr0.05153142
ELECTRON MICROSCOPYf_plane_restr0.00493424
ELECTRON MICROSCOPYf_dihedral_angle_d20.09782709

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