Simulated tempering yields insight into the low-resolution Rosetta scoring functions.

Rosetta is a structure prediction package that has been employed successfully in numerous protein design and other applications.1 Previous reports have attributed the current limitations of the Rosetta de novo structure prediction algorithm to inadequate sampling, particularly during the low-resolution phase.2-5 Here, we implement the Simulated Tempering (ST) sampling algorithm67 in Rosetta to address this issue. ST is intended to yield canonical sampling by inducing a random walk in temperatures space such that broad sampling is achieved at high temperatures and detailed exploration of local free energy minima is achieved at low temperatures. ST should therefore visit basins in accordance with their free energies rather than their energies and achieve more global sampling than the localized scheme currently implemented in Rosetta. However, we find that ST does not improve structure prediction with Rosetta. To understand why, we carried out a detailed analysis of the low-resolution scoring functions and find that they do not provide a strong bias towards the native state. In addition, we find that both ST and standard Rosetta runs started from the native state are biased away from the native state. Although the low-resolution scoring functions could be improved, we propose that working entirely at full-atom resolution is now possible and may be a better option due to superior native-state discrimination at full-atom resolution. Such an approach will require more attention to the kinetics of convergence, however, as functions capable of native state discrimination are not necessarily capable of rapidly guiding non-native conformations to the native state.