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- PDB-8rk2: Human Replication protein A (RPA; trimeric core) - ssDNA complex -

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Basic information

Entry
Database: PDB / ID: 8rk2
TitleHuman Replication protein A (RPA; trimeric core) - ssDNA complex
Components
  • Replication protein A 14 kDa subunit
  • Replication protein A 32 kDa subunit
  • Replication protein A 70 kDa DNA-binding subunit, N-terminally processed
KeywordsDNA BINDING PROTEIN / ssDNA binding protein / DNA damage repair / Single-strand annealing
Function / homology
Function and homology information


protein localization to chromosome / DNA replication factor A complex / single-stranded telomeric DNA binding / chromatin-protein adaptor activity / regulation of DNA damage checkpoint / Removal of the Flap Intermediate / protein localization to site of double-strand break / G-rich strand telomeric DNA binding / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) ...protein localization to chromosome / DNA replication factor A complex / single-stranded telomeric DNA binding / chromatin-protein adaptor activity / regulation of DNA damage checkpoint / Removal of the Flap Intermediate / protein localization to site of double-strand break / G-rich strand telomeric DNA binding / 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 / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / regulation of double-strand break repair via homologous recombination / telomeric DNA binding / site of DNA damage / Presynaptic phase of homologous DNA pairing and strand exchange / HSF1 activation / Activation of the pre-replicative complex / telomere maintenance via telomerase / PCNA-Dependent Long Patch Base Excision Repair / Regulation of HSF1-mediated heat shock response / mismatch repair / Activation of ATR in response to replication stress / SUMOylation of DNA damage response and repair proteins / mitotic G1 DNA damage checkpoint signaling / regulation of mitotic cell cycle / telomere maintenance / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic cell cycle / nucleotide-excision repair / Fanconi Anemia Pathway / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / double-strand break repair via homologous recombination / Translesion Synthesis by POLH / base-excision repair / G2/M DNA damage checkpoint / PML body / HDR through Homologous Recombination (HRR) / Meiotic recombination / Dual Incision in GG-NER / Formation of Incision Complex in GG-NER / DNA-templated DNA replication / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / single-stranded DNA binding / site of double-strand break / regulation of cell population proliferation / Processing of DNA double-strand break ends / protein phosphatase binding / DNA recombination / DNA replication / Regulation of TP53 Activity through Phosphorylation / damaged DNA binding / chromosome, telomeric region / nuclear body / DNA repair / ubiquitin protein ligase binding / DNA damage response / chromatin / enzyme binding / nucleoplasm / nucleus / metal ion binding
Similarity search - Function
Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain ...Replication factor A protein 2 / Replication protein A, C-terminal / Replication protein A C terminal / Replication factor A protein 3 / Replication factor A protein 3 / Replication factor-A protein 1, N-terminal domain / Replication factor A protein-like / Replication factor A protein 1 / Replication factor-A protein 1, N-terminal / Replication protein A, OB domain / Replication protein A OB domain / : / Replication factor A, C-terminal / Replication factor-A C terminal domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Replication protein A 32 kDa subunit / Replication protein A 70 kDa DNA-binding subunit / Replication protein A 14 kDa subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiang, C.C. / West, S.C.
Funding support United Kingdom, European Union, Switzerland, 8items
OrganizationGrant numberCountry
The Francis Crick InstituteCC2098 United Kingdom
Cancer Research UKCC2098 United Kingdom
Medical Research Council (MRC, United Kingdom)CC2098 United Kingdom
Wellcome TrustCC2098 United Kingdom
European Research Council (ERC)ERC-ADG-666400European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/W01355X/1 United Kingdom
Louis-Jeantet Foundation Switzerland
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nature / Year: 2024
Title: Mechanism of single-stranded DNA annealing by RAD52-RPA complex.
Authors: Chih-Chao Liang / Luke A Greenhough / Laura Masino / Sarah Maslen / Ilirjana Bajrami / Marcel Tuppi / Mark Skehel / Ian A Taylor / Stephen C West /
Abstract: RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of ...RAD52 is important for the repair of DNA double-stranded breaks, mitotic DNA synthesis and alternative telomere length maintenance. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA) and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal, and aberrant expression of RAD52 is associated with poor cancer prognosis. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.
History
DepositionDec 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 29, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replication protein A 70 kDa DNA-binding subunit, N-terminally processed
B: Replication protein A 32 kDa subunit
C: Replication protein A 14 kDa subunit


Theoretical massNumber of molelcules
Total (without water)111,0733
Polymers111,0733
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Replication protein A 70 kDa DNA-binding subunit, N-terminally processed


Mass: 68212.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA1, REPA1, RPA70 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P27694
#2: Protein Replication protein A 32 kDa subunit / RP-A p32 / Replication factor A protein 2 / RF-A protein 2 / Replication protein A 34 kDa subunit / RP-A p34


Mass: 29276.795 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA2, REPA2, RPA32, RPA34 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P15927
#3: Protein Replication protein A 14 kDa subunit / RP-A p14 / Replication factor A protein 3 / RF-A protein 3


Mass: 13583.714 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPA3, REPA3, RPA14 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P35244

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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: Open ring conformation of human RAD52 in complex with ssDNA
Type: COMPLEX
Details: Recombinant RAD52 purified from E. coli, and the open ring conformation was separated by cation exchange. The RAD52-ssDNA complex was reconstituted in vitro.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMSodium chlorideNaCl1
30.25 mMTCEPC9H15O6P1
40.1 mMCHAPSOC32H58N2O8S1
52 mMMagnesium acetateMg(CH3COO)21
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 46 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM software
IDNameCategory
1crYOLOparticle selection
2Topazparticle selection
5CTFFINDCTF correction
8UCSF ChimeraXmodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 21697126
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1316062 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 138.23 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00253307
ELECTRON MICROSCOPYf_angle_d0.44514466
ELECTRON MICROSCOPYf_chiral_restr0.0451499
ELECTRON MICROSCOPYf_plane_restr0.0036578
ELECTRON MICROSCOPYf_dihedral_angle_d4.3039437

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