(a) State occurrence of 5-fold symmetric (assigned to Closed), dome-shaped (assigned to Open-II), elevated bean-shaped (assigned to Open-I) and other asymmetric CorAs (unassigned, Open +) at different Mg2+-concentrations: 10 mM Mg2+: green, 3 mM Mg2+: blue, 0 mM Mg2+: red, and after re-addition of 25 mM Mg2+: yellow. Bars represent the normalized percentages of state assignments of ~20 CorA molecules in ~80–100 frames, ie up to ~2400 molecular representations, for each Mg2+-condition. Error bars are standard error of mean (s.e.m.). Below, schematic representations of the various conformations. (b) CorA state transition-maps at 10 mM, 3 mM, 0 mM, Mg2+ and after subsequent re-addition of Mg2+ to 25 mM (from left to right). The schematic molecule on the left (rows) is the state in frame(n) and the schematic molecule on the top (columns) is the state in frame(n+1). Numbers are normalized percentages of the state transitions of the same experimental data as in (a). Color scale was adapted for each condition separately with a gradient from green (lowest occurrence of transition) over yellow and orange to red (highest occurrence of transition). Numbers in the center of boxes of 4 state transitions represent the sum of transitions between states with elevated subunits (blue dashed square) and between transitions of strongly elongated structures (red square). (c) CorA conformational transition model based on the HS-AFM observations. Within ~10 min of Mg2+-depletion, the 5-fold symmetric, fully Mg2+-liganded CorA transit into dynamically fluctuating molecules with flexible subunits until their conformation stabilizes in a Mg2+-free highly asymmetric structure with increased membrane protrusion height. Figure 5 - Information Supplement 1: Estimation of thermally activated TM1 helix motions We estimated the theoretical range of helical motion by considering that TM1 behaves like a flexible rod undergoing thermally excited motions. The helix (rod) is characterized by a specific persistence length LP that is related to the bending stiffness KS through . The basic description for the change in curvature between two points on the rod is given by , with s being the arc length and a unit tangent vector at position (s). In an ideal system, the total elastic energy Eela of a particular conformation is given by the integral of the bending energies accumulated along a rod with contour length L: Assuming only circular curvatures along the rod, , where r is the radius of curvature. Using this basic description of an elastic polymer rod and considering the persistence length of protein α-helices Lp = 100 nm (as described in Choe and Sun, 2005) and a contour length L = 11 nm (length of TM1, see Supplementary Figure 4), we obtain This equation thus estimates that the radius of curvature r = ~23 nm at 1 kBT. Helix bending of that range would result in ~2 nm movements at its end.