Extended Data Fig. 1: EMC:CaV1.2(ΔC)/CaVβ3 and CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 Cryo-EM analysis.
From: EMC chaperone–CaV structure reveals an ion channel assembly intermediate
a-d, Exemplars of purified CaV1.2(ΔC)/CaVβ3: a, SEC (Superose 6 Increase 10/300 GL). b, peak fraction SDS-PAGE. c, electron micrograph (~105,000x magnification), and d, 2D class averages. e–h Exemplars of purified CaV1.2(ΔC)/CaVβ3/CaVα2δ-1: e, SEC (Superose 6 Increase 10/300 GL). f, peak fraction SDS-PAGE. Magenta bars in ‘a’ and ‘e’ mark peak fraction. g, electron micrographs (~105,000x magnification), and h, 2D class averages. i, Workflow for electron microscopy data processing for the CaV1.2(ΔC)/CaVβ3 and CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 samples. Initial cryoSPARC-3.2 Ab initio reconstruction identified a population of particles containing the EMC:CaV1.2(ΔC)/CaVβ3 complex in the CaV1.2(ΔC)/CaVβ3 sample and populations of particles containing either the EMC:CaV1.2(ΔC)/CaVβ3 complex or CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 complex in the CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 sample. Red arrows indicate the three classes that were re-extracted, subjected to multiple rounds of 3D heterogeneous classification, and exported from cryoSPARC-3.2 for further 3D refinement in RELION-3.1. This resulted in two maps for the EMC:CaV1.2(ΔC)/CaVβ3 complex (ECAB Maps 1 and 2) and one for the CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 complex (CABAD Map 1). Multibody refinement was performed in RELION-3.1 to improve the features of flexible regions of the three maps. This resulted in the final map for the CaV1.2(ΔC)/CaVβ3/CaVα2δ-1 complex (CABAD Map 2). ECAB Maps 1-2 with improved flexible features were merged (cross correlation = 0.9836) to obtain the final map for the EMC:CaV1.2(ΔC)/CaVβ3 complex (ECAB Map 3). Red boxes indicate the final maps used for model building. For ‘b-c’, N = 3. For ‘f-g’, N = 2.
