2.3. Metadynamics Simulation Parameters

Well-tempered metadynamics simulations were performed by Gromacs software patched by PLUMED 2.4.2 [65]. One of the challenges in metadynamics is the determination of the correct collective variables (CV). Since interactions between Ca²⁺ ions and carboxyl groups play a key role in structure formation, we chose the number of the carboxyl groups free from adsorbed Ca²⁺ ions as the collective variable. Free carboxyl groups were defined via:

where r is the distance between the carboxyl oxygen and a Ca²⁺ ion. The variable r₀ is defined as the maximum of the radial distribution function between the oxygens of carboxyl groups and a Ca²⁺ ion (the radial distribution function is shown in Figure S3). If the function is equal to zero, the carboxyl group is considered to be free from Ca²⁺ ions. Metadynamics is based on the addition of Gaussians to the energy landscape using CVs. Here, the Gaussian width was set to 0.05 and the initial Gaussian deposition rate to 2 kJ/mol per ps, with a bias factor of 20. The changing of CV during the time simulation is shown in Figure S4. We have equilibrated for an additional 150 ns using metadynamics and used the time interval 150–450 ns as a production run for collecting data. A similar approach has been successfully used to simulate ion adsorption on phospholipid membranes [66] and the formation of calcium phosphate prenucleation clusters [67].