Supplementary Figure 1: Activation mechanisms and 5-HT_2BR–methylergonovine structure.

a, Additional 5-HT_2BR ergoline SAR showing G_q-mediated calcium flux by N(1)-H-containing ergolines, LSD (red, EC₅₀ = 26 nM, E_max = 82%) and ergotamine (ERG, black, EC₅₀ = 334 nM, E_max = 85%) and antagonism by N(1)-alkyl ergolines, including LY215840, which contains N(1)-isopropyl (blue, IC₅₀ = 2.0 nM). Data are expressed as percent 5-HT response and represent means ± s.e.m. from two independent experiments (n = 2) performed in triplicate. b, Alignment of the 5-HT_2BR ERG (gray), LSD (purple) and methylergonovine (light blue) crystal structures reveals slight differences in positioning of the indole N(1)-H with respect to TM5. c, 5-HT_2BR–methylergonovine structure F_o – F_c omit map of ligand (left), 2F_o – F_c regular map of ligand (middle) and binding pocket residues (right). d, Surface expression measured by ELISA reveals similar expression levels of the T140^3.37 and A225^5.46 mutants as for wild-type 5-HT_2BR. Data represent means ± s.e.m. from quadruplicate replicates from two independent experiments (n = 2). e, F_o – F_c omit density map of residues T140^3.37 and L362^7.35 in the 5-HT_2BR–methylergonovine structure. f, 5-HT G_q-mediated calcium flux agonist potency at T140A^3.37 (red, EC₅₀ = 73 nM) and T140V^3.37 (blue, EC₅₀ = 93 nM) compared to T140S^3.37 (green, EC₅₀ = 5.7 nM) and wild-type 5-HT_2BR (black, EC₅₀ = 5.2 nM). Data are expressed as fold-over-basal and represent means ± s.e.m. from two independent experiments (n = 2) performed in triplicate. g, 5-HT G_q-mediated calcium flux agonist potency at A225S^5.46 (red, EC₅₀ = 1.4 nM) and A225G^5.46 (blue, EC₅₀ = 1.5 nM) as compared to wild-type 5-HT_2BR (black, EC₅₀ = 3.2 nM). Data are expressed as fold-over-basal and represent means ± s.e.m. from two independent experiments (n = 2) performed in triplicate.