Extended Data Fig. 6: 3D variability analysis of the cryo-EM data reveals different levels of flexibility in the engagement of the peptide in different activation states.

a, Frame #0 of PC1 reveals density for ECL2 but is absent in frame #19 of PC1. The greater degree of flexibility in ECL2 in the absence of bound peptide is consistent with its role in peptide binding as the side chains of residues T199^ECL2-F204^5x28 undergo flipping upon engagement with both the agonist and antagonist peptides (Extended Data Fig. 5). The flexible ECL2 is connected to TM4 that shows a rocking motion. b, Frame #0 of PC2 and PC3 reveals density for the C-terminal region of the antagonist, but no density for the N-terminal portion. Frame #19 of PC2 and PC3 reveals density for both the N-terminal and C-terminal regions. c, Comparison of start and end frames of PC1 in Ste2^IL•Ag reveals strong density for both the N-terminal and C-terminal regions of α-factor, but higher conformational flexibility in the middle of the peptide. d, Comparison of start and end frames of PC1 in Ste2^AL•Ag reveals strong density for the entire α-factor indicating a stronger engagement with the receptor. Density maps are displayed as transparent surface representation (Ste2_A, blue; Ste2_B green; ECL2, red; agonist (ago) and antagonist (ant) peptides, purple; ECL3, orange). The displayed density maps (surface representations) represent the indicated frames obtained from the 3D variability analysis procedure. The atomic model is from the respective cryo-EM structures that has been docked into the map frames for better visualization.