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- PDB-8g3g: CryoEM structure of yeast recombination mediator Rad52 -

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Basic information

Entry
Database: PDB / ID: 8g3g
TitleCryoEM structure of yeast recombination mediator Rad52
ComponentsDNA repair and recombination protein RAD52
KeywordsRECOMBINATION / Recombination mediator protein / DNA repair / Apo structure / Decamer
Function / homology
Function and homology information


HDR through Single Strand Annealing (SSA) / double-strand break repair via single-strand annealing / meiotic joint molecule formation / DNA amplification / DNA/DNA annealing activity / DNA recombinase assembly / SUMOylation of DNA damage response and repair proteins / mitotic recombination / DNA strand exchange activity / telomere maintenance via recombination ...HDR through Single Strand Annealing (SSA) / double-strand break repair via single-strand annealing / meiotic joint molecule formation / DNA amplification / DNA/DNA annealing activity / DNA recombinase assembly / SUMOylation of DNA damage response and repair proteins / mitotic recombination / DNA strand exchange activity / telomere maintenance via recombination / double-strand break repair via break-induced replication / DNA damage tolerance / mitochondrial DNA repair / nuclear chromosome / double-strand break repair via homologous recombination / mitochondrion / 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 and recombination protein RAD52
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsDeveryshetty, J. / Basore, K. / Rau, M. / Fitzpatrick, J.A.J. / Antony, E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM133967, GM130746 United States
CitationJournal: Nat Commun / Year: 2023
Title: Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.
Authors: Jaigeeth Deveryshetty / Rahul Chadda / Jenna R Mattice / Simrithaa Karunakaran / Michael J Rau / Katherine Basore / Nilisha Pokhrel / Noah Englander / James A J Fitzpatrick / Brian Bothner / Edwin Antony /
Abstract: Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher ...Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.
History
DepositionFeb 7, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
J: DNA repair and recombination protein RAD52
A: DNA repair and recombination protein RAD52
B: DNA repair and recombination protein RAD52
C: DNA repair and recombination protein RAD52
D: DNA repair and recombination protein RAD52
E: DNA repair and recombination protein RAD52
F: DNA repair and recombination protein RAD52
G: DNA repair and recombination protein RAD52
H: DNA repair and recombination protein RAD52
I: DNA repair and recombination protein RAD52


Theoretical massNumber of molelcules
Total (without water)524,76510
Polymers524,76510
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Sedimentation velocity Mass Photometer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
DNA repair and recombination protein RAD52


Mass: 52476.496 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: RAD52, YML032C / Plasmid: pTXB1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P06778

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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: Rad52 decamer / Type: COMPLEX
Details: Full length yeast Rad52 expressed with chitin binding domain (CBD) tag. CBD was removed by thiol induced cleavage by intein.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 520 kDa/nm / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Cellular location: Nucleus
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) / Plasmid: pTXB1
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acidHEPES1
2100 mMSodium ChlorideNaCl1
31 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 97 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 150000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50.7 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.2.0particle selection
2EPUimage acquisition
4cryoSPARC3.2.0CTF correctionPatch CTF estimation
7Coot0.9.3model fitting
11cryoSPARC3.2.0classificationheterogeneous refinement
12cryoSPARC3.2.03D reconstruction
13PHENIXmodel refinementreal space refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 701962
SymmetryPoint symmetry: C10 (10 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46845 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Initial model was built de novo in ModelAngelo tool. the best-looking subunit was real space refined in Phenix, followed by manual building in Coot. Later C10 symmetry was applied, and real ...Details: Initial model was built de novo in ModelAngelo tool. the best-looking subunit was real space refined in Phenix, followed by manual building in Coot. Later C10 symmetry was applied, and real space refined in Phenix.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047120
ELECTRON MICROSCOPYf_angle_d0.7789570
ELECTRON MICROSCOPYf_dihedral_angle_d4.881000
ELECTRON MICROSCOPYf_chiral_restr0.0451060
ELECTRON MICROSCOPYf_plane_restr0.0051240

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