Refinement via MD simulations was more successful at higher temperatures (Figure ). The refinement performance improved as MD simulation temperature increased up until 360 K before decreasing at higher temperatures, especially for GDT-HA scores. Refinement at higher temperatures is expected to be effective for overcoming energy barriers, so that conformational transitions would be more likely to occur and a wider range of conformations can be sampled. This is indeed what we find (see example in Figure ). At moderately increased temperatures (i.e. 360 K), the additional sampling includes more native-like structures (Figure ) but when the temperature is too high (i.e. 380 K) sampling shifts to non-native states. Moreover, while sampling based on simulations at 298.15 K resulted in sampled conformations that were well packed with low RWplus scores, the conformations shifted to less-well packed structures at 380K with higher RWplus scores even although secondary structure arrangements were overall improved (Figure ). At 360 K, structures were overall improved and retained favorable RWplus scores indicative of good packing resulting in the best refinement predictions after application of the post-sampling stage. To understand the effect of temperature in more detail, we compared the refinement performance consistency at different temperatures. (Figure ). Refinement results can vary for independent runs because of stochastic sampling. Sampled conformational space can be different between independent sets of simulations as we use limited sampling for refinement. Based on such an analysis, we find that refinement at higher temperatures is more consistent, meaning that different refinement runs for the same target produce more similar results than at lower temperatures. Presumably, this is because it was easier to escape from local minima at higher temperatures. On the other hand, at lower temperatures, it took more time to get over local minima, so sampled conformational space could be different between independent refinement simulations as they were kinetically trapped in local minima. The consequence of this observation is that fewer refinement runs, at less computational costs, may be sufficient when using elevated temperatures to achieve reliable refinement.