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- PDB-7ste: Rad24-RFC ADP state -

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Basic information

Entry
Database: PDB / ID: 7ste
TitleRad24-RFC ADP state
Components
  • (Replication factor C subunit ...) x 4
  • Checkpoint protein RAD24
KeywordsREPLICATION / DNA damage / DNA replication / DNA sliding clamp
Function / homology
Function and homology information


DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / DNA clamp loader activity / Translesion Synthesis by POLH / Translesion synthesis by REV1 ...DNA clamp unloading / Rad17 RFC-like complex / Gap-filling DNA repair synthesis and ligation in GG-NER / Elg1 RFC-like complex / Ctf18 RFC-like complex / DNA replication factor C complex / Polymerase switching / DNA clamp loader activity / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / mitotic DNA replication checkpoint signaling / reciprocal meiotic recombination / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / mismatch repair / DNA damage checkpoint signaling / nucleotide-excision repair / DNA-templated DNA replication / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / 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 / : / DNA polymerase III, delta subunit / : ...Rad17 P-loop domain / Checkpoint protein Rad17/Rad24 / Checkpoint protein Rad17/Rad24, fungi/metazoa / 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 / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Checkpoint protein RAD24 / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsCastaneda, J.C. / Schrecker, M. / Remus, D. / Hite, R.K.
Funding support3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107239
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127428
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Mechanisms of loading and release of the 9-1-1 checkpoint clamp.
Authors: Juan C Castaneda / Marina Schrecker / Dirk Remus / Richard K Hite /
Abstract: Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, ...Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.
History
DepositionNov 12, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jun 5, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Replication factor C subunit 4
C: Replication factor C subunit 3
D: Replication factor C subunit 2
E: Replication factor C subunit 5
A: Checkpoint protein RAD24
hetero molecules


Theoretical massNumber of molelcules
Total (without water)236,48211
Polymers234,2415
Non-polymers2,2406
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Replication factor C subunit ... , 4 types, 4 molecules BCDE

#1: Protein Replication factor C subunit 4 / Replication factor C4 / Activator 1 37 kDa subunit


Mass: 36201.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RFC4, YOL094C, O0923 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339
#2: Protein Replication factor C subunit 3 / Replication factor C3 / Activator 1 40 kDa subunit


Mass: 38155.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RFC3, YNL290W, N0533 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629
#3: Protein Replication factor C subunit 2 / Replication factor C2 / Activator 1 41 kDa subunit


Mass: 39794.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RFC2, YJR068W, J1808 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348
#4: Protein Replication factor C subunit 5 / Replication factor C5 / Activator 1 40 kDa subunit


Mass: 39993.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RFC5, YBR087W, YBR0810 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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Protein , 1 types, 1 molecules A

#5: Protein Checkpoint protein RAD24


Mass: 80096.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: RAD24, YER173W, SYGP-ORF60 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32641

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Non-polymers , 3 types, 6 molecules

#6: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#7: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Rad24-RFC:9-1-1:DNA / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
125 mMHepes-KOH1
2300 mMKOAc1
37 mMMg(OAc)21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4432412
3D reconstructionResolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 183334 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11SXJ

1sxj

11SXJ1PDBexperimental model
23A1J

3a1j

13A1J2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00214488
ELECTRON MICROSCOPYf_angle_d0.4719588
ELECTRON MICROSCOPYf_dihedral_angle_d4.2751960
ELECTRON MICROSCOPYf_chiral_restr0.0372252
ELECTRON MICROSCOPYf_plane_restr0.0032476

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