However, the initial model for refinement usually lies off the protein folding pathway and direct transition to the native state is hindered by significant kinetic barriers, e.g. due to mispacked side chains. In essence, refinement often requires that structures are partially unfolded and refolded. These transitions demand a long time due to kinetic barriers. From a systematic analysis of energy landscapes between initial homology models and the native states it was found that effective time scales for crossing the kinetic barriers during refinement may be on the order of a few microseconds to sub-milliseconds for small proteins and probably even longer for larger systems. Very extensive MD simulations and/or the application of enhanced sampling methods such as replica exchange MD simulation have been proposed to overcome the sampling challenge. However, such protocols can be technically challenging and may require significant computational resources that limit their practical use in routine applications of refinement methods. Therefore, standard MD simulations have remained the most effective sampling approach for exploring diverse conformational states with limited computational expense and complexity.