(A) Ion flux measurements of purified Orai mutants. K+ flux measurements for Orai with the H206A, P288L, or V174A mutations under divalent-free conditions. The assay was performed as previously described (Hou et al., 2018; Hou et al., 2012). Proteoliposomes containing purified Orai with the indicated mutations were loaded with 150 mM KCl and were diluted 50-fold into flux buffer containing a fluorescent pH indicator (ACMA) and 150 mM N-methyl-D-glucamine (NMDG) to establish a K+ gradient (Materials and methods). After stabilization of the fluorescence signal (150 s), a proton ionophore (CCCP) was added. An electric potential arising from K+ efflux drives the uptake of protons, which quenches the fluorescence of ACMA. The time-dependent decrease in fluorescence observed for the H206A and V174A after the addition of CCCP is indicative of K+ flux, whereas K+ flux is not detected for the P288L mutant. Valinomycin, which renders the vesicles permeable to K+, was added near the end of the experiment (black arrows) as a positive control and to establish baseline fluorescence. Traces were normalized by dividing by the initial fluorescence value, which was within ±10% for each experiment. The experiments shown used a protein-to-lipid ratio of 1:100 (wt/wt). Higher concentrations of the P288L mutant (ratios of 1:10 and 1:50) yielded analogous results; ion flux was not detected through P288L Orai. As expected, wild-type Orai does not show K+ flux in this assay as it is not active without STIM (Hou et al., 2018; Hou et al., 2012). (B) Superposition of an X-ray structure of P288L Orai (Liu et al., 2019) with an X-ray structure of Orai without this mutation (WT Orai) in the unlatched-closed conformation, in which the pore is closed (Hou et al., 2018). Two opposing subunits from each structure are shown, with the WT and P288L structures depicted as blue and magenta ribbons, respectively. The RMSD of the superposition is 1.5 Å (for Cα atoms from M1 to M4). (C) Superposition of the electron densities. Electron densities for WT and P288L Orai are drawn in blue and magenta mesh representations, respectively, covering the two subunits shown in (B). The map of WT Orai (contoured at 1.3 σ) was calculated from 20 to 6.9 Å resolution using amplitudes and MR-SAD phases that had been improved by NCS averaging, solvent flattening, and histogram matching (as described in Hou et al., 2018). The density from the P288L Orai structure (2Fo-Fc electron density map, contoured at 1.5 σ) was generated using map coefficients FWT and PHWT downloaded from RCSB Protein Data Bank (PDB ID: 6AKI). The structure and electron density superpositions show that the two structures are indistinguishable within the transmembrane region of the channel and that both structures contain the narrow hydrophobic region characteristic of a closed pore. An asterisk indicates the density assigned to an anion/iron complex that was observed in the basic region of the closed pore (Hou et al., 2018; Hou et al., 2012). Similar density is present in the P288L structure, but this density was modeled as Cl− (Liu et al., 2019). However, because Cl− ions would not be visible in low-resolution maps, we suggest that the density more likely represents the anion/iron complex that is observed when the pore is closed (Hou et al., 2018; Hou et al., 2012).