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- EMDB-41357: Cryo-EM structure of human full-length RAD52 -

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Basic information

Entry
Database: EMDB / ID: EMD-41357
TitleCryo-EM structure of human full-length RAD52
Map dataSharpened map used for modeling
Sample
  • Complex: Human RAD52 undecameric structure
    • Protein or peptide: DNA repair protein RAD52 homolog
KeywordsDNA repair / RECOMBINATION
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 / HDR through MMEJ (alt-NHEJ) / regulation of nucleotide-excision repair / 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 / HDR through MMEJ (alt-NHEJ) / regulation of nucleotide-excision repair / 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: 2.5 Å
AuthorsSchnicker NJ / Razzaghi M / Spies M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA232425-05 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131704-05 United States
CitationJournal: Nature / Year: 2025
Title: The RAD52 double-ring remodels replication forks restricting fork reversal.
Authors: Masayoshi Honda / Mortezaali Razzaghi / Paras Gaur / Eva Malacaria / Giorgia Marozzi / Ludovica Di Biagi / Francesca Antonella Aiello / Emeleeta A Paintsil / Andrew J Stanfield / Bailey J ...Authors: Masayoshi Honda / Mortezaali Razzaghi / Paras Gaur / Eva Malacaria / Giorgia Marozzi / Ludovica Di Biagi / Francesca Antonella Aiello / Emeleeta A Paintsil / Andrew J Stanfield / Bailey J Deppe / Lokesh Gakhar / Nicholas J Schnicker / M Ashley Spies / Pietro Pichierri / Maria Spies /
Abstract: Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation. ...Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses, we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the single-stranded DNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.
History
DepositionJul 25, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41357.png

Downloads & links

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Map

FileDownload / File: emd_41357.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used for modeling
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 247.65 Å		0.83 Å/pix.
x 300 pix.
= 247.65 Å		0.83 Å/pix.
x 300 pix.
= 247.65 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8255 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.0017248477 - 1.7862712
Average (Standard dev.)0.0020899153 (±0.033294037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.65001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map 2

Fileemd_41357_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_41357_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human RAD52 undecameric structure

EntireName: Human RAD52 undecameric structure
Components
  • Complex: Human RAD52 undecameric structure
    • Protein or peptide: DNA repair protein RAD52 homolog

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Supramolecule #1: Human RAD52 undecameric structure

SupramoleculeName: Human RAD52 undecameric structure / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 532 KDa

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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: 48.40402 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGH RVINLANEMF GYNGWAHSIT QQNVDFVDLN NGKFYVGVCA FVRVQLKDGS YHEDVGYGVS EGLKSKALSL E KARKEAVT ...String:
MGSSHHHHHH SSGLVPRGSH MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGH RVINLANEMF GYNGWAHSIT QQNVDFVDLN NGKFYVGVCA FVRVQLKDGS YHEDVGYGVS EGLKSKALSL E KARKEAVT DGLKRALRSF GNALGNCILD KDYLRSLNKL PRQLPLEVDL TKAKRQDLEP SVEEARYNSC RPNMALGHPQ LQ QVTSPSR PSHAVIPADQ DCSSRSLSSS AVESEATHQR KLRQKQLQQQ FRERMEKQQV RVSTPSAEKS EAAPPAPPVT HST PVTVSE PLLEKDFLAG VTQELIKTLE DNSEKWAVTP DAGDGVVKPS SRADPAQTSD TLALNNQMVT QNRTPHSVCH QKPQ AKSGS WDLQTYSADQ RTTGNWESHR 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

Concentration0.25 mg/mL
BufferpH: 9
Component:
ConcentrationNameFormula
20.0 mMTris
200.0 mMPotassium chlorideKCl
1.0 mMDTT

Details: 20 mM Tris pH 9, 200 mM KCl, 1 mM DTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Details: -15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was made from frozen protein

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Electron microscopy

MicroscopeTFS KRIOS
DetailsNo tilt data and tilted data (30 deg) was collected at the same time and processed together
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 11288 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
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: 2261059
Details: Template based picking from no tilt and 30 deg tilt data to deal with preferred orientation
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1kn0

Final reconstructionApplied symmetry - Point group: C11 (11 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 623559
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1kn0


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial fitting was done in Chimera followed by refinement with Namdinator and then PHENIX alone
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 130.2 / Target criteria: Cross-correlation coefficient
Output model

PDB-8tkq:
Cryo-EM structure of human full-length RAD52

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