Extended Data Fig. 4: Cryo-EM map quality of the Ste2–mini-Gpa1–Ste4–Ste18 heterotrimer complex and model validation.

Unless stated otherwise, densities from map 1 were visualized using UCSF ChimeraX³⁵ (contour level 0.03) and encompass a carve radius of 2 Å around the indicated region. a, Densities of transmembrane helices of Ste2_A (contour level 0.045), α-factor_B, mini-Gpa1_B, dimer interface between Ste2_A and Ste2_B, intracellular loops ICL1–3 and extracellular loops ECL1–3 are shown as a mesh. Densities of αN and α4–β6 are shown as representative densities from mini-Gpa1_A. Densities of Ste4 and Ste18 (contour 0.025) are shown as a mesh at a contour level of 0.01 and a carve radius of 2 Å. b, Two N-acetylglucosamine molecules were modelled attached to Asn25 of Ste_A and Ste2_B and they contribute to the dimer interface (contour level 0.01 from map 2). Asn25 is a well-characterized glycosylation site in Ste2¹⁷. c, Densities for six ordered putative CHS molecules surrounding the dimer interface were visible and were modelled (contour level 0.01). Several other detergent or lipid acyl-chain densities were visible in the map before detergent micelle signal subtraction, but were left unmodelled. d, FSC of the refined model versus the map (green curve) and FSC_work/FSC_free validation curves (orange and blue curves, respectively).