Principal component and mutual information analysis of global conformational changes induced by PIP2 unbinding. (A) Projection of the hTPC2 conformations onto the first two eigenvectors of a PCA of all trajectories in the set, showing a separation of the states in this subspace (super-open PIP2-bound state, green; apo-closed state, blue; open state as in the cryo-EM structure [non-conductive], cyan). (B) Time evolution of the projection on eigenvector 1 for all trajectories; color code as in A. (C) State-specific mutual information (SSI) analysis of the allosteric coupling between the PIP2 binding site, the bundle crossing, and the SF (color spectrum from blue, 0.11 bits, to red, 0.93 bits of mutual information). The location of the residues coupled with the ligand status indicates the formation of allosteric pathways between the ligand binding site, the bundle crossing, and the SF (magenta arrows, right). Key residues in the SF are labeled; important conformational switches in the SF, the bundle crossing region, as well as the ligand are shown in ball-and-stick representation. The values are symmetric between the subunits; only one subunit is shown in the color code for clarity.