Fig. 5.

Limited change in EFSAM secondary structure after Ca²⁺ depletion. a–c Far-UV CD spectra of WT EFSAM-GrpE, EFSAM-2NQ-GrpE, and EFSAM(D76A)-GrpE, each protein 5 μM, in the absence or presence of Ca²⁺. d–g Examples of deuterium uptake into four peptic peptides of EFSAM WT, D76A and 2NQ in Ca²⁺-free and Ca²⁺-bound forms, at 0.5, 1, 2 and 5 min time points, determined by HDX–MS. h Deuterium exchange into EFSAM peptides of wild-type, D76A, and 2NQ proteins, in 0 Ca²⁺ or 30 μM Ca²⁺, at the 0.5 min time point. The peptides from d–g are marked with arrowheads. Supplementary Fig. 9b presents the data for all time points. i Differences in deuterium exchange between the inactive and active forms of wild-type EFSAM (WT(Ca²⁺-bound)–WT(Ca²⁺-free)), between the active form of wild-type EFSAM and the D76A protein (WT(Ca²⁺-free)–D76A(Ca²⁺-free)), and between the inactive form of wild-type EFSAM and the 2NQ protein (WT(Ca²⁺-bound)–2NQ(Ca²⁺-bound)), at the 0.5 min time point. Data are the averages of triplicate measurements. j Spatial positions of the low-exchanging peptide (EVIQWLIT; green) and two flanking higher-exchanging peptides (WKAWKSSEVYNWTVDE and YVELPQYEET; orange) highlighted on the Ca²⁺-bound WT EFSAM structure (PDB ID: 2K60)