Extended Data Fig. 4: Workflow for obtaining the cryo-EM structures of the RAD52 double undecamer bound to synthetic fork DNA.

a. For single particle analysis, micrographs were manually curated and multiple rounds of 2D classification performed yielding a final stack of 63,576 particles of 2D classes containing RAD52 double rings. The selected 2D class averages were used as templates followed by multiple rounds of 2D classification yielding 124,131 particles prior to ab-initio reconstruction, heterogeneous refinement (4 classes) and homogeneous refinement of final structures of RAD52-fork DNA. The heatmaps of the angular distribution of particles used to generate the final RAD52-fork DNA structures (EMD-42066 and EMD-42069) are shown. The templates were then created from final maps and used for template picking and particle extraction from micrographs (1,945,503 particles) followed by multiple rounds of 2D classification and heterogeneous refinement. The final double-ring structure of EMD-42440 was obtained by performing non-uniform refinement, 2D classification, ab-initio reconstruction, and homogeneous refinement. b-d. The heatmaps and resolution evaluation for the RAD52-fork structures. b. The heatmap of the angular distribution of particles used to generate the final RAD52-fork DNA structures are shown. c. GSFSC curves from cryoSPARC are shown for the final RAD52-fork DNA structures. d. FSC curves with and without mask were calculated using Mtriage as part of Phenix package.