Simulating FRET Efficiency in Clathrin-Mediated Endocytosis

FRET simulations (Supplementary Figs. 4c and 18) were performed using MATLAB. We used 60 Å for Förster radius for EGFP-ShadowY FRET. For the simulation in Supplementary Fig. 4c, we determined the position of N- and C-terminus of CLC according to the structure models (PDB 3LVG and 6WCJ)^{17 (link),46 (link)}. For the simulation in Supplementary Fig. 18, the lateral position of N-terminus was moved by 0.6 Å spacing, and N-terminus was assumed to locate axially 25 Å higher than C-terminus. When we focus on single clathrin heavy chain, there are four different states; without CLC, endogenous CLCa or CLCb binding, EGFP-CLC binding, or CLC-ShadowY binding (Supplementary Fig. 4b). By considering these four states, we calculated the FRET efficiency between EGFP-CLC and CLC-ShadowY or ShadowY-CLC binding to surrounding five clathrin heavy chains at various heavy chain occupancy with ShadowY-attached CLC. $\mathrm{Occupancy}=\frac{\left[\mathrm{ShadowY}\mathrm{probe}\mathrm{binding}\right]}{\left[\mathrm{No}\mathrm{CLC}\mathrm{binding}\right]+\left[\mathrm{Endogenous}\mathrm{CLCa}/\mathrm{b}\mathrm{biding}\right]+\left[\mathrm{EGFP}-\mathrm{CLC}\mathrm{binding}\right]+\left[\mathrm{ShadowY}\mathrm{probe}\mathrm{binding}\right]}$