Uncertainties in Markov State Models of Small Proteins

Markov state models are widely used to describe and analyze protein dynamics based on molecular dynamics simulations, specifically to extract functionally relevant characteristic time scales and motions. Particularly for larger biomolecules such as proteins, however, insufficient sampling is a notorious concern and often the source of large uncertainties that are difficult to quantify. Furthermore, there are several other sources of uncertainty, such as choice of the number of Markov states and lag time, choice and parameters of dimension reduction preprocessing step, and uncertainty due to the limited number of observed transitions; the latter is often estimated via a Bayesian approach. Here, we quantified and ranked all of these uncertainties for four small globular test proteins. We found that the largest uncertainty is due to insufficient sampling and initially increases with the total trajectory length T up to a critical tipping point, after which it decreases as 1/T, thus providing guidelines for how much sampling is required for given accuracy. We also found that single long trajectories yielded better sampling accuracy than many shorter trajectories starting from the same structure. In comparison, the remaining sources of the above uncertainties are generally smaller by a factor of about 5, rendering them less of a concern but certainly not negligible. Importantly, the Bayes uncertainty, commonly used as the only uncertainty estimate, captures only a relatively small part of the true uncertainty, which is thus often drastically underestimated.