Bootstrapping/subsampling was used to investigate if using a larger frame spacing could still lead to reproducible results for these systems. This would also practically mean a reduced number of frames to analyze (e.g., a 0.4 ns spacing bootstrap calculation has half the number of frames as a 0.2 ns calculation). The results obtained for these bootstrapping with replacement and random subsampling calculations for both PTP1B and the PDZ3 domain are presented in Figures 9B and S16B. Our results show that a reduced rate of sampling could indeed be used as the per residue scores would still be reproducible. Naturally, there are limits to how large the sampling rate can be which is most clearly demonstrated for the PDZ3 domain (Figures and). That said, it would appear reasonable that for both test systems, a reduced sampling rate of 0.6 ns would be enough to obtain reliable and reproducible results (when performed alongside using 5 pairs of replicas).