(A) Cartoon representation of the components of guanylyl cyclase C (GC-C) signaling and Hsp90–Cdc37 regulation and the zippered and activated GC-C. GC-C is colored in red, guanylin/uroguanylin (Gn/Uro) in yellow, Hsp90 in blue and teal, and Cdc37 in purple. Extracellular domains (ECD), transmembrane domain (TM), pseudokinase domain (PK), dimerization domain (DD), and guanylyl cyclase domain (GC) are labeled. In the rightmost cartoon, the regions unobserved in the cryo-EM density are in a lighter shade with a dashed outline. (B) The refined and sharpened cryo-EM density map of GC-C–Hsp90–Cdc37, colored as in A, with a transparent overlay of an unsharpened map with additional DD density resolved. Cdc37 coil-coiled and middle domain (MD) are labeled. (C) Reference-free 2D averages for the GC-C–Hsp90–Cdc37 complex. (D) The refined and sharpened cryo-EM density map of GC-C–Hsp90–Cdc37, colored as in A and B, labeled with all domains as in A and B, with the addition of Hsp90 N-terminal domain (NTD), middle domain (MD), and C-terminal domain (CTD). (E) Ribbon representation of a model of GC-C–Hsp90–Cdc37 complex, colored and labeled as in A, B, and C.