Extended Data Fig. 2: AA150µM-TAS2R14-Gg-scFv16, FFA1mM-TAS2R14-miniGs/gust-Nb35 and FFA350µM -TAS2R14-Gi1-scFv16 sample preparation, cryo-EM data processing and structure determination.
From: Bitter taste TAS2R14 activation by intracellular tastants and cholesterol
(a, f, k) Representative size exclusion chromatography (SEC) profiles, SDS-PAGE analysis, representative cryo-EM micrography (scale bar: 50 nm) and selected 2D classification averages of AA150µM-TAS2R14-Gg-scFv16 (a), FFA1mM-TAS2R14-miniGs/gust-Nb35 (f) and FFA350µM -TAS2R14-Gi1-scFv16 (k) complexes. (b, g, l) Cryo-EM data processing flowcharts of AA150µM-TAS2R14-Gg-scFv16 (b), FFA1mM-TAS2R14-miniGs/gust-Nb35 (g) and FFA350µM -TAS2R14-Gi1-scFv16 (l) complexes by cryoSPARC 4.3. (c, h, m) Cryo-EM maps are colored by local resolution (Å) for AA150µM-TAS2R14-Gg-scFv16 (c), FFA1mM-TAS2R14-miniGs/gust-Nb35 (h) and FFA350µM -TAS2R14-Gi1-scFv16 (m) complexes. (d, i, n) Angular distribution of the particles used for final reconstruction (up) and ‘Gold-standard’ FSC curve at the FSC = 0.143 (down) for AA150µM-TAS2R14-Gg-scFv16 (d), FFA1mM-TAS2R14-miniGs/gust-Nb35 (i) and FFA350µM -TAS2R14-Gi1-scFv16 (n) complexes. (e, j, o) The density maps for helices TM1-TM7 of transmembrane domain, helix 8 and α5 helices of Gα for AA150µM-TAS2R14-Gg-scFv16 (e), FFA1mM-TAS2R14-miniGs/gust-Nb35 (j) and FFA350µM -TAS2R14-Gi1-scFv16 (o) complexes. The SEC and SDS-PAGE experiments were repeated at least three times with similar results.