Fig. 7

Mechanistic events enabling opening of the pathway for lipid flip from the IC to the EC leaflet. a Two-dimensional landscape representing mapping of all Stage 3 trajectories of the WT nhTMEM16 in POPC membrane (WT^ensemble) onto the first two tICA eigenvectors (tICA 1 and tICA 2, see 'Methods' for details of tICA analysis). The location of microstates 1–6 that represent the translocation of the lipid through nhTMEM16 groove is denoted and the structural representation of these microstates are given in the surrounding snapshots. In these structural models, the advancing lipid is rendered in licorice (colored according to atom type), and relevant groove residues are shown in space fill representations and labeled (in microstates 2 and 5). Microstate denoted by * captures rare mode of protein–lipid interaction in which the hydrocarbon tail of a lipid inserts into the EC side of the groove interfering with the translocation of the advancing lipid (see also Supplementary Figure 10c). b Sequence of mechanistic events leading to the lipid flip. c Structural characteristics of the selected six microstates. The columns from left to right record the probability distributions of Z-directional distance between the phosphorus atom of the translocated lipid and the Cα atom of R432 (the 0 on the x-axis represents position of the lipid with its phosphorus atom aligned in the Z direction with the Cα atom of R432), of lipid tilt angle, of minimum distance between T333 and Y439, and of opening between the EC ends of TM3 and TM6 helices measured as the distance between the center-of-masses of two groups: C_α atoms of residues 315 to 318 on TM3, and C_α atoms of residues 432 to 435 on TM6