The crystal structure of wild-type Channelrhodopsin 2 (PDB ID: 6EID 5 ) was used as a starting point. The dimeric form of ChR2 was generated in VMD using the BIOMT transformation matrix written in the PDB file. A protein preparation workflow was followed as implemented in Maestro 27 to take it from its raw state to a state where the protein is properly prepared for MD calculations. First, co-crystallized molecules and phosphate ions were removed, and missing atoms from some residues were added. The hydrogen bonding network was optimized by reorienting hydroxyl and thiol groups, water molecules, the amide groups of asparagine (Asn) and glutamine (Gln), and the imidazole rings of histidine (His). The protonation states of His, aspartic acid (Asp) and glutamic acid (Glu) were predicted based on the local environment of each residue at pH 7.4 as determined from a pKa prediction by the PROPKA3 method. Consequently, the RSB, as well as the Asp, Glu, Lys, and Arg residues, were modeled in their ionic form in both chains, except for Asp156, Glu90, and Glu101, which were protonated. All His residues were modeled in their neutral state, with the proton in either N ϵ (residues 114, 134, 201, 265 in chain A and residues 134, 191, 201, 265 in chain B) or in N δ (residues 191, 249, 272, 274, 278 in chain A and residues 114, 249, 272, 274, 278 in chain B). Finally, a minimization is performed with the OPLS4 force field with a threshold criterion being an RMSD below 0.3 Å for the heavy atoms. After this minimization, disulfide bonds between cysteins (Cys) were formed between Cys36 (chain A) and Cys34 (chain B), and between Cys34 (chain A) and Cys36 (chain B). The CHARMM-GUI membrane builder was used to embed the protein in a 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) bilayer solvated by water and a concentration of 0.15 M NaCl. The box size of the system is 120 nm × 120 × 151 nm.