Extended Data Fig. 2: Cryo-EM analysis of the human Cav3.1-Δ8b alone and in complex with Z944.
From: Cryo-EM structures of apo and antagonist-bound human Cav3.1
a, Whole-cell patch clamp measurements of the full-length human Cav3.1 and Cav3.1-Δ8b. n values indicate the number of independent cells; mean ± s.e.m. b, Last-step purification of human Cav3.1-Δ8b. Shown here is a representative size-exclusion chromatogram for proteins obtained from 30 l of HEK293F cells transfected with plasmids. The indicated peak fractions on the Coomassie-blue-stained SDS–PAGE (Supplementary Fig. 2) were pooled and concentrated for cryo-EM sample preparation. c, Representative electron micrograph and 2D class averages. The green circles indicate representative particles in distinct orientations. The black and white scale bars in the top and bottom panels represent 100 nm and 10 nm, respectively. d, Flowchart for EM data processing. Details can be found in Methods. e, The gold-standard Fourier shell correlation (FSC) curves for the 3D reconstructions. The middle and right panels show FSC curves for phase-randomized half maps and unmasked half maps for the apo (middle) and complex (right) datasets. f, FSC curves of the refined model versus the overall map that it was refined against (black); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (red); and of the model refined in the first of the two independent maps versus the second independent map (green). The small difference between the red and green curves indicates that the refinement of the atomic coordinates did not suffer from overfitting. Before calculation of FSC against model-generated map, both half maps and the merged map were multiplied by a solvent mask that only includes the protein region. The merged map was brought to a threshold at which the micelle is invisible and all transmembrane helices are visible. Dust points were manually removed using the hide dust function in Chimera. Caution was taken not to mask out the densities for the bound ligand and lipids. The map was then extended by 2 pixels and supplied with a soft edge width of 12 pixels using relion_mask_create. g, Local-resolution map for the 3D EM reconstruction of Cav3.1-Δ8b in the presence of Z944. The map, calculated in RELION-3.1, was generated in Chimera49.
