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- EMDB-19253: Human RAD52 open ring - ssDNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-19253
TitleHuman RAD52 open ring - ssDNA complex
Map dataSharpen by DeepEMhancer
Sample
  • Complex: Open ring conformation of human RAD52 in complex with ssDNA
    • Protein or peptide: DNA repair protein RAD52 homolog
    • DNA: ssDNA
  • Ligand: MAGNESIUM ION
  • Ligand: water
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: 2.3 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 21, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19253.png

Downloads & links

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Map

FileDownload / File: emd_19253.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpen by DeepEMhancer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å		0.85 Å/pix.
x 360 pix.
= 306. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0191
Minimum - Maximum-0.0018199041 - 1.7122481
Average (Standard dev.)0.0005731121 (±0.015978409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 306.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpen by -71.5 B Factor

Fileemd_19253_additional_1.map
AnnotationSharpen by -71.5 B Factor
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_19253_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19253_half_map_2.map
Projections & Slices
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Sample components

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Entire : Open ring conformation of human RAD52 in complex with ssDNA

EntireName: Open ring conformation of human RAD52 in complex with ssDNA
Components
  • Complex: Open ring conformation of human RAD52 in complex with ssDNA
    • Protein or peptide: DNA repair protein RAD52 homolog
    • DNA: ssDNA
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Open ring conformation of human RAD52 in complex with ssDNA

SupramoleculeName: Open ring conformation of human RAD52 in complex with ssDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
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.
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: 9 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.232656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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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Macromolecule #2: ssDNA

MacromoleculeName: ssDNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.951477 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 39 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.15 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
0.25 mMC9H15O6PTCEP
0.5 %C14H28O6Octyl beta glucoside
2.0 mMMg(CH3COO)2Magnesium acetate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 13176589
Startup modelType of model: INSILICO MODEL / In silico model: RELION 3D initial model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 587046
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8rj3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8rjw:
Human RAD52 open ring - ssDNA complex

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