Fig. 5.

Investigation of the DBD-A–DBD-E interaction. a A proposed model for the interaction between Rfa1-DBD-A (blue) and Rfa3 (green) based upon the interaction interface between Ustilago maydis DBD-A and Rfa3 in the crystal structure asymmetric unit. The model was created using the yeast DBD-A NMR structure (pdb 1ynx) and the refined Rfa3 model from our cryo-EM data, superposed onto DBD-A and Rfa3 of pdb 4gop. Residues R78, L88, and R60 that we mutate in this study are located at the interface and are illustrated by yellow spheres. A Mec1-dependent DBD-A phosphorylation site (S178) close to the interaction interface is labeled. b–e Microscale thermophoresis (MST)-binding curves using purified DBD-A versus fluorescently labeled Rfa3-OB domain (residues 1–99) together with binding curves for the Rfa3-OB point mutants (substitutions given above curves). Normalized fluorescence was calculated using NanoTemper Analysis 1.2.101 and is plotted as function of DBD-A concentration. The apparent effective half maximal binding (EC₅₀) was calculated by fitting using the hill equation in Prism and is given for each binding experiment together with the calculated maximum fluorescence (Bmax) and R² of the fit. n.d. denotes not determined. f Bovine serum albumin (BSA) control for non-specific binding is also shown. Each experiment was performed in triplicate with each data point an average with standard error of the mean (SEM) shown. Source data are provided as a Source Data file