Fig. 1

EFSAM-GrpE design and Ca²⁺ responsiveness. a Cartoon of activated STIM1 (58-473) as inferred from the literature⁴⁵. Domain organization is marked. Residues 24–57 and 473–685 are not depicted. b Cartoon of the expected EFSAM-GrpE structure used in the current study, showing structural similarity to the extended activated STIM1. Green ovals represent EFSAM (58-209) and blue cartoon denotes Thermus thermophilus GrpE. GrpE is not structurally related to STIM1 except for the presence of extended α-helices that form a coiled coil. The coiled coil is constitutive in GrpE, unlike in STIM1. c Schematic of the EFSAM-GrpE construct design. d Size exclusion chromatography of the Ca²⁺-bound (20 mM Ca²⁺; blue line) and Ca²⁺-free (5 mM EGTA; red line) forms of EFSAM-GrpE. e Schematic of chemical labelling of EFSAM-GrpE, depicting the case where individual monomers are labelled with fluorescein and AF594. Other possible combinations in the random labelling approach used here are not illustrated. f Fluorescence emission spectra (λ_ex = 420 nm) of single-cysteine EFSAM-GrpE labelled with fluorescein (Donor alone), AF594 (Acceptor alone) or with both dyes (Donor and Acceptor). All measurements were made in Ca²⁺-free buffer. g Fluorescence spectral scans (λ_ex = 420 nm) of double-labelled EFSAM-GrpE with varying amounts of Ca²⁺