Fig. 3.

Arrangement of the calcium-binding residues elucidated by cryo-EM and MD. (A) Overlay of two adjacent subunits of DeCLIC cryo-EM structures with and without Ca²⁺ (aquamarine and purple, respectively). Protein domains and main Ca²⁺-binding motifs are shown. Those include the transmembrane domain (TMD), extracellular domain (ECD), and two amino-terminal domains (NTD1 and NTD2) as well as the β1 to β2 loop from the principal subunit (P) and loop F from the complementary subunit (C). The boxed-in view (Bottom) shows the relevant side chains (sticks, colored by heteroatom). The Ca²⁺ ion present in the Ca²⁺ dataset (aquamarine) is colored in yellow. (B) Cryo-EM panels (Left) represent the cryo-EM structures and their corresponding densities (mesh at α  =  0.012 to 0.015). Relevant side chains around the Ca²⁺-binding site [loop F (C), β1 to β2 loop (P)] are displayed as sticks (heteroatom coloring). The density for the Ca²⁺-binding residues as well as for the Ca²⁺ ion (in the with-Ca²⁺ condition) is well resolved. MD panels (Right) illustrate eleven individual conformations (one snapshot for every 100 ns) from 1-μs long MD simulations of the cryo-EM models. The relevant Ca²⁺ residues are displayed as sticks (colored by heteroatom). In cyan, we have Na⁺ ions (sphere, trajectories generated for every 10 ns) and Ca²⁺ ion in yellow. In the no-Ca²⁺ condition, the Na⁺ ions are free to enter the cavity which is occupied by Ca²⁺ in the with-Ca²⁺ condition.