(1-100 ns), and both pulling and US simulations were performed without any orthogonal restraints. This is because our main goal for this system was to reproduce and assess the US protocol published by Lemkul and co-workers more than 10 years ago. This protocol is also used in widely spread GROMACS tutorials, which is the starting point for many users in the area of MD simulations and US. With much faster pulling, the projection of initial structures onto the reaction coordinate is not uniformly distributed (see Figure ), compared to systems that were pulled more slowly (Figures ). When pulled fast, the system can sample only a small portion of configuration space, which leads to larger differences between pathways, as compared to slower pulling.(Figure , S21). However, differences between pathways remain.