Fig. 3: Structural characterization.

a Representative 2D class averages of integrin with and without minibinder. For αvβ6, both closed and open headpiece conformations are present in the unbound state, but in the presence of the minibinder the open conformation is dominant. For αvβ8, no open headpieces were observed, with or without minibinder. b CryoEM density map of αvβ6 bound to minibinder B6_BP_dslf. B6_BP_dslf (goldenrod) binds the integrin ligand binding cleft between the αv (green) and β6 (light blue) subunits and induces or stabilizes the open conformation. The sharpened, locally refined cryoEM map is shown in color, superimposed with the unsharpened map showing all domains of the αvβ6 headpiece in semi-transparent white. c, d Overlay of the designed αvβ6 + B6_BP_dslf model (gray) and the experimentally determined cryoEM model (colors). Although the overall angle of the minibinder is shifted, the RGD loop positioning is as predicted. Insets in c and d are magnified in panels h and i, respectively. e CryoEM density map of αvβ8 bound to minibinder B8_BP_dslf. Similar to B6_BP_dslf, B8_BP_dslf (brown) binds the integrin ligand binding cleft between the αv (green) and β8 (blue) subunits, however the conformation of αvβ8 remains in the closed headpiece conformation. The sharpened, locally refined cryoEM map is shown in color, superimposed with the unsharpened map showing all domains in the αvβ8 ectodomain construct in semi-transparent white. f, g An overlay of the designed αvβ8 + B8_BP_dslf model (gray) and the experimentally determined model. Although the overall angle of the minibinder is shifted, the RGD loop positioning is as predicted. Insets in f and g are magnified in panels i and k, respectively. h, i Key designed interactions between β-loop and β6/β8 subunit are observed in the cryoEM structure: K41 from B6_BP_dslf forms a salt bridge with E316 from the β6 subunit (panel h). E41 from β-loop makes backbone level hydrogen bond with I216 from β8 subunit and D40 makes salt bridge interaction with K304 from β8 subunit (panel i). j Experimental vs designed (gray) packing pattern of the LXXL motif and SDL2 of αvβ6. k Experimental vs designed (gray) packing pattern of the MAVY motif and SDL2 of αvβ8.