AbstractAccurate modeling of biomolecular systems requires accurate forcefields. Widely used molecular mechanics (MM) forcefields obtain parameters from experimental data and quantum chemistry calculations on small molecules but do not have a clear way to take advantage of the information in high‐resolution macromolecular structures. In contrast, knowledge‐based methods largely ignore the physical chemistry of interatomic interactions, and instead derive parameters almost exclusively from macromolecular structures. This can involve considerable double counting of the same physical interactions. Here, we describe a method for forcefield improvement that combines the strengths of the two approaches. We use this method to improve the Rosetta all‐atom forcefield, in which the total energy is expressed as the sum of terms representing different physical interactions as in MM forcefields and the parameters are tuned to reproduce the properties of macromolecular structures. To resolve inaccuracies resulting from possible double counting of interactions, we compare distribution functions from low‐energy modeled structures to those from crystal structures. The structural and physical bases of the deviations between the modeled and reference structures are identified and used to guide forcefield improvements. We describe improvements resolving double counting between backbone hydrogen bond interactions and Lennard‐Jones interactions in helices; between sidechain‐backbone hydrogen bonds and the backbone torsion potential; and between the sidechain torsion potential and Lennard‐Jones interactions. Discrepancies between computed and observed distributions are also used to guide the incorporation of an explicit Cα‐hydrogen bond in β sheets. The method can be used generally to integrate different sources of information for forcefield improvement. Proteins 2011; © 2011 Wiley‐Liss, Inc.