Fig. 3.

Ca²⁺ loop three is most important to SdrG B1 domain stability. a Equilibrated homology model of SdrG B1, with N- and C-terminal ß-strands marked in blue and red, respectively. Ca²⁺ coordinating amino-acid side chains shown as sticks. Ca²⁺ binding sites one to three are marked (Ca1 purple, Ca2 cyan, Ca3 orange). A conserved glutamine bridge (light gray) connects the Ca1 loop to Ca3. Amino acids mutated to remove respective loops are underlined in the sequence shown. b Closeup of each Ca²⁺ binding loop, including non-sidechain coordinating residues in stick representation. c, d Comparison of absolute unfolding forces of mutated SdrG B1 in 10 mM EDTA (red) and 10 mM Ca²⁺ (green) with a single cantilever, also given in percentage of the WT strong state. Errors are the full-width at half maximum of the BE fits for each unfolding force distribution (see Supplementary Fig. 6), underlying raw force datapoints are shown as black horizontal lines. The single loop knockouts in (c) show that Ca3 is crucial for overall stability and most likely remains bound in the weak state. Once Ca3 is removed, weak state forces drop to only 6–8% of WT strength. Removing Ca1 or Ca2 or both (d) supports this as the weak state remains at 31% of WT. The glutamine bridge (Ca1QKO) seems to be of minor importance for the overall domain stability