(A) The secondary structure of the erCry4 model obtained after extensive MD equilibration, with the isoalloxazine part of the FAD (red) and the conserved tryptophan triad (magenta).

(B) Superimposed molecular structures of the FAD isoalloxazine part and the tryptophan triad from atCry1 (blue), tetrad from dmCry1 (orange), and the tetrad from erCry4 model (red).

(C) Structural alignment of atCry1 (blue), dmCry1 (orange), and the erCry4 model (red), suggesting two major structural differences in the three proteins: (1) the surface-exposed parts of the proteins feature one large helix in atCry1, whereas dmCry1 and the erCry4 model feature three and two smaller helices respectively (blue box), and (2) atCry1 and dmCry1 feature one helix at the end of the PHR domain, while the erCry4 model features a shorter helix in the same structural area (green box). One reason for this discrepancy might be that dmCry1 is the only cryptochrome where the C terminus is resolved in the crystal structure, whereas the atCry1 structure and the erCry4 model only include the PHR domain. To include the complete structure of atCry1 and erCry4 would change the C-terminal part, which, unfortunately, cannot be predicted reliably yet.

(D and E) Comparison of the amino acid residues surrounding the FAD in the binding pocket of atCry1 (D) and dmCry1 and erCry4 (D and E).

(F) Homology models of proteins obtained directly from web servers can rarely be used as reliable structures for further analysis since these models are usually not stabilized. The stability of the homology model can be established and probed by MD simulations, as done here for the model of erCry4. (F) shows the root-mean-square deviation (RMSD) of the protein backbone atoms computed relatively to the initial structure of the model for erCry4 during the entire 0.5-μs MD simulation performed on our erCry4 model. The gray area indicates the equilibration interval, while the remaining simulation was used to justify the stability of the model (production simulation).