Supplementary Materials

The PDF file includes:

• Fig. S1. NFAT activation by constitutively active STIM1 mutants.
• Fig. S2. Constitutive CAD dissociation for the STIM1-H72R mutant.
• Fig. S3. Ca²⁺ ion binding to the EF-SAM domain.
• Fig. S4. Ca²⁺-binding probability and RMSD values for the STIM1 canonical EF-hand mutants.
• Fig. S5. Altered protein unfolding for the STIM1-F108I mutant.
• Legends for movies S1 and S2

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Other Supplementary Material for this manuscript includes the following:

• Movie S1 (.mov format). Multiple Ca²⁺-binding events to the EF-hand domains of STIM1.
• Movie S2 (.mov format). A single bound Ca²⁺ ion maintains the luminal STIM1 structure.