Thus tight coupling of Ca2+ activation and ATP hydrolysis is assured. Although full convergence cannot be rigorously demonstrated from a single trajectory, our simulations are far longer than any previous ones in this system, the trajectories appear to be stable, and they are in good agreement with complementary FRET measurements. Thus these simulations appear to capture essential features of the intrinsic dynamics of SERCA activation. Each of the two free energy landscapes constructed here is based on a single 500 ns simulation, so the results should be interpreted with caution �C the free energy wells would probably deepen significantly if the simulations were extended. However, the free energy landscape and collective motions combine to suggest that Ca2+ splits the ensemble of open Ginkgolide-C conformations into two minima to selectively produce the collective motions of the N domain necessary for the closure of the headpiece toward the active conformation. Considering that SERCA populates open, partially open and closed conformations in solution, and based on comparison of the free energy landscapes of both Apo and bound SERCA, we suggest that the open conformation of SERCA belongs to a conformational pathway necessary for the selective activation of the high Ca2+ affinity conformation of SERCA by Ca2+. This goes beyond previous observations, not only suggesting that the open conformation of SERCA can exist in solution, but also that the open conformation is a functional element of the conformational space of the pump. A particularly important region of the free energy landscape of Ca2+ bound SERCA is region 3, which corresponds to the actual open-to-closed transition. Unlike Apo SERCA, this region of the energy landscape is very rugged, and is characterized by multiple local minima separated by small energy barriers. This indicates that Ca2+ binding reshapes the free energy landscape to contain several Trifluridine kinetic traps. We suggest that these kinetic traps are an intrinsic feature of Ca2+ bound SERCA, and that they act as checkpoints to drive the headpiece through a well-defined pathway toward a correct geometry necessary for cphosphate transfer to Asp351. This suggestion also challenges the current view that the orientational distribution of the N domain could represent a random sampling of conformations in solution. While this is probably true for Apo SERCA, our results strongly suggest that in the presence of Ca2+ the N domain strictly follows a well-defined pathway between the open and closed conformations. This exquisite malleability of the free energy landscape appears responsible to fine-tune SERCA activity: in Apo SERCA, the energy landscape ensures that ATP will not be used unnecessarily, while in the presence of Ca2+ the reshaping of the landscape guarantees that SERCA is not trapped in an unproductive conformation, therefore facilitating ATP hydrolysis and the formation of the phosphoenzyme complex, necessary for calcium transport to the lumen. Although X-ray crystallography has proven to be a powerful tool in understanding the atomic structure of SERCA, this study supports the conclusion, based on previous FRET experiments, that the open crystal structure represents a local energy minimum that is not predominant in solution. It is clear that solution spectroscopic methods such as FRET, EPR, and NMR are needed to connect real-time protein dynamics with function, resolving transitions among multiple conformational states.