Extended Data Fig. 3. Stoichiometry and ligand binding of the Orco homotetramer.

a, Western blot of cross-linked Orco in transfected HEK293 cells. SDS-PAGE showing a ladder of four bands that appears with both GFP-tagged and untagged Orco after treatment with increasing concentration of the amine cross-linker disuccinimidyl suberate (DSS). Monomer (m) and tetramer (t) bands are indicated. DSS concentrations were (μM): 0, 25, 75, 125, 250, 2500. b, Western blot of a Blue Native (BN)-PAGE⁶³ gel of the same samples as in a showing that tetrameric Orco is present in all samples and higher-order aggregates are not induced by cross-linking. c,d Western blot of Orco extracted with increasing concentrations of detergent (LMNG) showed gradual loss of the tetrameric species. Addition of (c) CHS, (d) porcine brain polar lipid extract (BPL; Anatrace) or the combination of the two stabilized the Orco tetramer. LMNG concentrations were (% w/v): 0.01, 0.05, 0.1, 0.25, 0.5, 1. Concentration of CHS, BPL and the sum of CHS + BPL were added at 1/5 that of LMNG. GFP-labeled Orai (55 kDa)⁶⁴ was used as a molecular weight marker as it is a hexamer with a similar total size as the Orco-GFP tetramer (340 kDa). The larger apparent molecular weights observed in BN-PAGE gels (b–d) reflect the additional mass of the micelle. Primary antibodies used were: anti-Orco clone 20F7 and anti-GFP (Life Technologies). Each experiment in this figure was repeated three times with similar results. The molecular weight markers on the native gels are approximate: they are from a separate gel run under the same conditions (see Supplementary Data). e,f, Representative baseline-corrected isothermal titration calorimetry (ITC) data for (e) Orco (11 μM) and (f) Orco-Fab complex (10 μM) titrated with VUAA1. Integrated heats and fitted single-site binding isotherms are shown at the bottom. The number of binding sites per monomer was fixed at 1 and the dissociation constant (K_d), enthalpy of binding (ΔH), and heat associated with sample dilution (ΔQ_dil) were fit. The experiments were repeated three times each using Orco samples obtained from independent purifications. The average fitted thermodynamic parameters are as follows (mean ± s.d.). Orco: K_d = 13 ± 1 μM, ΔH = −8.3 ± 0.6 kcal/mol, ΔQ_dil = −1.0 ± 0.1 kcal/mol. Orco-Fab: K_d = 18 ± 2 μM, ΔH = −9.7 ± 0.4 kcal/mol, ΔQ_dil = −1.0 ± 0.2 kcal/mol.