Frame alignment was performed using MotionCor2 (31), and CTF (contrast transfer function) correction, using Gctf (32) for both data sets, was performed on non–dose-weighted micrographs, while subsequent data processing was performed on dose-weighted micrographs. For both data sets, manual picking for automatic picking template generation, automatic picking, and particle extraction were performed using Relion 2.0 (33), and all subsequent data processing was performed using cryoSPARC (34). For each data set, ~2000 particles were manually selected to generate 2D classes for use in automatic picking.

In processing the hTRPV6-CaM data set, six 2D classes were used as templates for automatically picking 429,167 particles from 4515 micrographs. An ab initio map generated by cryoSPARC from a random selection from the entire pool of particles served as a reference for 3D classification (heterogeneous refinement) into three classes with no symmetry imposed. Ab initio map generation and 3D classification were performed iteratively a total of three times, each time selecting the best of three classes from the previous step as an initial pool for ab initio model generation. Eventually, a pool of 180,600 particles making up the best class from the third round of classification was subjected to homogeneous refinement, which resulted in a 3.98 Å resolution reconstruction. The rTRPV6-CaM data set was processed using a workflow similar to that described above. A total of 274,507 particles were picked and extracted from 1447 micrographs. Ab initio map generation for rTRPV6-CaM was not successful, most likely because of poor angular distribution of particle views in the initial random pool compared to hTRPV6-CaM. Instead, we used the density for hTRPV6-CaM as a reference for rTRPV6-CaM 3D classifications. After three consecutive rounds of 3D classification, a pool of 111,593 particles was subjected to homogeneous refinement, which generated a 3.60 Å resolution reconstruction. No symmetry was ever applied throughout the processing of either the hTRPV6-CaM or the rTRPV6-CaM data sets. The Fourier shell correlation (FSC) and local resolutions were calculated using cryoSPARC, and EM density maps were visualized using UCSF Chimera (35).

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