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- EMDB-29695: CryoEM structure of yeast recombination mediator Rad52 -

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Basic information

Entry
Database: EMDB / ID: EMD-29695
TitleCryoEM structure of yeast recombination mediator Rad52
Map data
Sample
  • Complex: Rad52 decamer
    • Protein or peptide: DNA repair and recombination protein RAD52
KeywordsRecombination mediator protein / DNA repair / Apo structure / Decamer / RECOMBINATION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsDeveryshetty J / Basore K / Rau M / Fitzpatrick JAJ / Antony E
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29695.png

Downloads & links

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Map

FileDownload / File: emd_29695.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.94 Å/pix.
x 300 pix.
= 282. Å		0.94 Å/pix.
x 300 pix.
= 282. Å		0.94 Å/pix.
x 300 pix.
= 282. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.94 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 7.9
Minimum - Maximum-26.399206 - 59.779198000000001
Average (Standard dev.)-0.000000000003631 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 282.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_29695_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29695_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rad52 decamer

EntireName: Rad52 decamer
Components
  • Complex: Rad52 decamer
    • Protein or peptide: DNA repair and recombination protein RAD52

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Supramolecule #1: Rad52 decamer

SupramoleculeName: Rad52 decamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Full length yeast Rad52 expressed with chitin binding domain (CBD) tag. CBD was removed by thiol induced cleavage by intein.
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Location in cell: Nucleus
Molecular weightTheoretical: 520 kDa/nm

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Macromolecule #1: DNA repair and recombination protein RAD52

MacromoleculeName: DNA repair and recombination protein RAD52 / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Molecular weightTheoretical: 52.476496 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNEIMDMDEK KPVFGNHSED IQTKLDKKLG PEYISKRVGF GTSRIAYIEG WRVINLANQI FGYNGWSTEV KSVVIDFLDE RQGKFSIGC TAIVRVTLTS GTYREDIGYG TVENERRKPA AFERAKKSAV TDALKRSLRG FGNALGNCLY DKDFLAKIDK V KFDPPDFD ...String:
MNEIMDMDEK KPVFGNHSED IQTKLDKKLG PEYISKRVGF GTSRIAYIEG WRVINLANQI FGYNGWSTEV KSVVIDFLDE RQGKFSIGC TAIVRVTLTS GTYREDIGYG TVENERRKPA AFERAKKSAV TDALKRSLRG FGNALGNCLY DKDFLAKIDK V KFDPPDFD ENNLFRPTDE ISESSRTNTL HENQEQQQYP NKRRQLTKVT NTNPDSTKNL VKIENTVSRG TPMMAAPAEA NS KNSSNKD TDLKSLDASK QDQDDLLDDS LMFSDDFQDD DLINMGNTNS NVLTTEKDPV VAKQSPTASS NPEAEQITFV TAK AATSVQ NERYIGEESI FDPKYQAQSI RHTVDQTTSK HIPASVLKDK TMTTARDSVY EKFAPKGKQL SMKNNDKELG PHML EGAGN QVPRETTPIK TNATAFPPAA APRFAPPSKV VHPNGNGAVP AVPQQRSTRR EVGRPKINPL HARKPT

UniProtKB: DNA repair and recombination protein RAD52

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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: 7.8
Component:
ConcentrationFormulaName
50.0 mMHEPESN-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
100.0 mMNaClSodium Chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 97 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.7 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 150000
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 701962
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C10 (10 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 46845
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 3.2.0) / Software - details: heterogeneous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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