To build models of the hTRPV6-CaM and rTRPV6-CaM complexes in COOT (36), we docked the structures of hTRPV6 [Protein Data Bank (PDB) ID: 6BO8], TRPV6* (PBD ID: 5WO7) (28), and CaM (PDB ID: 2L7L) (37) into the cryo-EM densities and used them as a guide. The resulting models were refined against unfiltered half maps in real space with constraints using PHENIX (38). The models were tested for overfitting (figs. S1 and S5) by shifting their coordinates by 0.5 Å (using shake) in PHENIX and generating densities in Chimera (35) from the shaken models. FSC was calculated between the densities from the shaken models, the half maps used in PHENIX refinement, the other half maps, and the unfiltered sum maps using EMAN2 (39). Structures were visualized, and figures were prepared in PyMOL (40).

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