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- EMDB-42069: Cryo-EM density map of a double-ring of human RAD52 in the presen... -

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Basic information

Entry
Database: EMDB / ID: EMD-42069
TitleCryo-EM density map of a double-ring of human RAD52 in the presence of fork DNA
Map dataunsharpenned EM density map from cryoSPARC's homogeneous refinement job
Sample
  • Complex: Cryo-EM density of full length human RAD52 DNA repair protein on a fork DNA
    • Protein or peptide: DNA repair protein RAD52 double-ring on a fork DNA
KeywordsDNA repiar protein / Recombination
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsRazzaghi M / Schnicker NJ / 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
DepositionSep 21, 2023-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42069.png

Downloads & links

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Map

FileDownload / File: emd_42069.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpenned EM density map from cryoSPARC's homogeneous refinement job
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 320 pix.
= 306.88 Å		0.96 Å/pix.
x 320 pix.
= 306.88 Å		0.96 Å/pix.
x 320 pix.
= 306.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.959 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.029
Minimum - Maximum-0.04758006 - 0.1529047
Average (Standard dev.)0.0026727216 (±0.012562483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 306.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A from cryoSPARC's homogeneous refienement job which is...

Fileemd_42069_half_map_1.map
Annotationhalf_map_A from cryoSPARC's homogeneous refienement job which is noisy and did gaussian filtering using cimerax command as: vop gaussian #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B from cryoSPARC's homogeneous refienement job which is...

Fileemd_42069_half_map_2.map
Annotationhalf_map_B from cryoSPARC's homogeneous refienement job which is noisy and did gaussian filtering using cimerax command as: vop gaussian #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 : Cryo-EM density of full length human RAD52 DNA repair protein on ...

EntireName: Cryo-EM density of full length human RAD52 DNA repair protein on a fork DNA
Components
  • Complex: Cryo-EM density of full length human RAD52 DNA repair protein on a fork DNA
    • Protein or peptide: DNA repair protein RAD52 double-ring on a fork DNA

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Supramolecule #1: Cryo-EM density of full length human RAD52 DNA repair protein on ...

SupramoleculeName: Cryo-EM density of full length human RAD52 DNA repair protein on a fork DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: RAD52 makes a double ring structure in the presence of fork DNA
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: DNA repair protein RAD52 double-ring on a fork DNA

MacromoleculeName: DNA repair protein RAD52 double-ring on a fork DNA / type: protein_or_peptide / ID: 1
Details: Cryo-EM density map of full length human RAD52 double ring on a fork DNA
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGHR VINLANEMFG YNGWAHSITQ QNVDFVDLNN GKFYVGVCAF VRVQLKDGSY HEDVGYGVSE GLKSKALSLE KARKEAVTDG ...String:
MGSSHHHHHH SSGLVPRGSH MSGTEEAILG GRDSHPAAGG GSVLCFGQCQ YTAEEYQAIQ KALRQRLGPE YISSRMAGGG QKVCYIEGHR VINLANEMFG YNGWAHSITQ QNVDFVDLNN GKFYVGVCAF VRVQLKDGSY HEDVGYGVSE GLKSKALSLE KARKEAVTDG LKRALRSFGN ALGNCILDKD YLRSLNKLPR QLPLEVDLTK AKRQDLEPSV EEARYNSCRP NMALGHPQLQ QVTSPSRPSH AVIPADQDCS SRSLSSSAVE SEATHQRKLR QKQLQQQFRE RMEKQQVRVS TPSAEKSEAA PPAPPVTHST PVTVSEPLLE KDFLAGVTQE LIKTLEDNSE KWAVTPDAGD GVVKPSSRAD PAQTSDTLAL NNQMVTQNRT PHSVCHQKPQ AKSGSWDLQT YSADQRTTGN WESHRKSQDM KKRKYDPS

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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

Concentration0.26 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
30.0 mMTris
75.0 mMKCl
5.0 mMMgCl2
1.0 mMDTT

Details: 30 mM TrisHCl pH 7.5, 75 mM KCl, 5 mM MgCl2, 1 mM DTT
GridSupport film - Material: CARBON / 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 and prepared fork DNA

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 2 / Number real images: 6093 / Average exposure time: 7.0 sec. / Average electron dose: 50.0 e/Å2 / Details: The images recorded from 2 grids.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 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: 1755381
Details: 2D classes containing double-rings used as a template for Template-based extraction of particles from 2 grids
CTF correctionSoftware - Name: cryoSPARC (ver. 4.2.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1)
Details: The global estimate of resolution is reported as 8.5A but the range spans from 6.6 to 16A.
Number images used: 33883
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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