Therefore, the biasing force is only applied to the system once a threshold of samples has been accumulated in each bin. In our simulations, the system evolves unbiased until 500 samples have been achieved in each bin, which has been found to be a solid trade-off between delayed ABF application and unwanted non-equilibrium results. From 500 up to 1000 samples, there is a linear correction factor for the ABF force from 0 to 1, while the ABF methodology is fully applied after 1000 samples are collected in each bin.