The starting structure used was based on the coordinates of a D. melanogaster cryptochrome crystal structure [Protein Data Bank (PDB) ID: 4GU5] (15, 22). The parameters for the oxidized and anion radical FAD were based on previous work (42). The simulations were run using GROMACS 5.0.4 (43) and the CHARMM27 force field. The simulation box was replicated in all directions with periodic boundary conditions, all bonds were constrained using the linear constraint solver algorithm, and a time step of 2 fs was used. Particle mesh Ewald (PME) electrostatics with fourth-order interpolation and with a grid spacing of 0.12 nm were used. The cutoff scheme was Verlet with a 1.0-nm cutoff, and cutoffs for short-range electrostatic and van der Waals interactions were 1.0 nm as well. During the production run, pressure control was achieved using the Parrinello-Rahman barostat (τP = 2 ps, P = 1 bar), and temperature control was achieved via the modified Berendsen (velocity-rescale) thermostat (τT = 0.1 ps, T = 300 K). The protein was placed in a cubic box, 1 nm larger than the protein in all directions and solvated with transferable interparticle potential with 3 points (TIP3P) water molecules. The system was neutralized by replacing water molecules with sodium and chloride ions to a final concentration of 150 mM. After adding ions, the system underwent initial energy minimization until all forces were below 1000 kJ mol−1 nm−1. Subsequently, the system was minimized for 100 ps in the canonical (NVT) and isothermal-isobaric (NPT) ensembles. During equilibration all nonhydrogen atoms were position restrained with force constants of 1000 kJ mol−1 nm−2. In total, 16 simulations were run for 250 ns each. These simulations used two different force-field parameters for the FAD, and in half of the simulations, histidine 378 was mutated to an alanine. In the simulations with H378 present, it was protonated on Nϵ since this is suggested to be the protonation state of H378 in the dark-adapted DmCry (20). Eight simulations each for the wild type and mutant were using the parameters for oxidized FAD and the anion radical, each with four replicates.

The RMSD of the FFW motif was used as a reporter of the CTT flexibility. Before computing the RMSD, rotational and translational degrees of freedom were removed by aligning the protein on the backbone atoms of residues that displayed the lowest RMSF. The average RMSF for all simulations was computed, and the protein was aligned on the quartile of the residues with the lowest RMSF. After this, the RMSD of the FFW motif was calculated.

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