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- EMDB-19189: Human RAD52 closed ring -

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Basic information

Entry
Database: EMDB / ID: EMD-19189
TitleHuman RAD52 closed ring
Map datahuman RAD52_closed_ring
Sample
  • Complex: Closed ring conformation of human RAD52
    • Protein or peptide: DNA repair protein RAD52 homolog
KeywordsssDNA binding protein / DNA damage repair / Single-strand annealing / DNA BINDING PROTEIN
Function / homology
Function and homology information


double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination ...double-strand break repair via single-strand annealing / DNA double-strand break processing involved in repair via single-strand annealing / DNA recombinase assembly / mitotic recombination / regulation of nucleotide-excision repair / HDR through MMEJ (alt-NHEJ) / HDR through Single Strand Annealing (SSA) / SUMOylation of DNA damage response and repair proteins / protein-DNA complex / double-strand break repair via homologous recombination / double-strand break repair / single-stranded DNA binding / cellular response to oxidative stress / DNA recombination / protein-containing complex / DNA binding / nucleoplasm / identical protein binding / nucleus
Similarity search - Function
DNA recombination/repair protein Rad52 / DNA repair protein Rad52/59/22 / Rad52 family / DNA repair protein Rad52/59/22 superfamily / Rad52/22 family double-strand break repair protein
Similarity search - Domain/homology
DNA repair protein RAD52 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiang CC / West SC
Funding support United Kingdom, European Union, Switzerland, 8 items
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 18, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19189.png

Downloads & links

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Map

FileDownload / File: emd_19189.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman RAD52_closed_ring
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.736
Minimum - Maximum-4.161617 - 7.3172784
Average (Standard dev.)0.0023902182 (±0.16003491)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 261.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: human RAD52 closed ring

Fileemd_19189_half_map_1.map
Annotationhuman RAD52_closed_ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: human RAD52 closed ring

Fileemd_19189_half_map_2.map
Annotationhuman RAD52_closed_ring
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Closed ring conformation of human RAD52

EntireName: Closed ring conformation of human RAD52
Components
  • Complex: Closed ring conformation of human RAD52
    • Protein or peptide: DNA repair protein RAD52 homolog

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Supramolecule #1: Closed ring conformation of human RAD52

SupramoleculeName: Closed ring conformation of human RAD52 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinant RAD52 purified from E. coli, and the closed ring conformation was separated by cation exchange.
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD52 homolog

MacromoleculeName: DNA repair protein RAD52 homolog / type: protein_or_peptide / ID: 1 / Number of copies: 11 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.232656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGHR VINLANEMFG YNGWAHSIT QQNVDFVDLN NGKFYVGVCA FVRVQLKDGS YHEDVGYGVS EGLKSKALSL EKARKEAVTD GLKRALRSFG N ALGNCILD ...String:
MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGHR VINLANEMFG YNGWAHSIT QQNVDFVDLN NGKFYVGVCA FVRVQLKDGS YHEDVGYGVS EGLKSKALSL EKARKEAVTD GLKRALRSFG N ALGNCILD KDYLRSLNKL PRQLPLEVDL TKAKRQDLEP SVEEARYNSC RPNMALGHPQ LQQVTSPSRP SHAVIPADQD CS SRSLSSS AVESEATHQR KLRQKQLQQQ FRERMEKQQV RVSTPSAEKS EAAPPAPPVT HSTPVTVSEP LLEKDFLAGV TQE LIKTLE DNSEKWAVTP DAGDGVVKPS SRADPAQTSD TLALNNQMVT QNRTPHSVCH QKPQAKSGSW DLQTYSADQR TTGN WESHR KSQDMKKRKY DPS

UniProtKB: DNA repair protein RAD52 homolog

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
0.25 mMC9H15O6PTCEP
5e-05 %C58H114O26Tween-20
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1908147
Startup modelType of model: INSILICO MODEL / In silico model: RELION 3D initial model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 309471
FSC plot (resolution estimation)

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