crystal structure of Batho (chain A of PDB ID 2G87). 1 ASP83 and GLU122 were considered to be neutral 2 while all other charged amino acids were set to default protonation states. The spatial positions of Batho with respect to the lipid bilayer were taken from the Orientations of Proteins in Membranes (OPM) database. 3 All histidines were protonated either in the N position (HIS65, HIS152, HIS195, and HIS278) or in the N δ position (HIS100 and HIS211) 4 . The Schiff base linkage between the retinal and LYS296 was positively charged. The retinal and all crystallographic water molecules were preserved, other heteroatoms were removed.The CHARMM-GUI web-server 5 was used to embed the Batho system in an explicit membrane bilayer, and then immerse the resulted protein-lipid system in a box of explicit waters and ions at physiological concentration. Two palmitoyl chains bound to CYS322 and CYS323 were added to Batho before it was inserted in the membrane, which consisted of 128 palmitoyl-oleoyl-phosphatidyl choline (POPC) lipids. The final system was neutral and the size of the periodic box was approximately 71×71×92Å 3 with 8589 water molecules, 20 sodium ions, and 18 chloride ions (see Fig. S6).Simulation Details All simulations were performed with the software suite GROMACS-5.1.1. 6The c36 CHARMM force field 7 was used for protein and lipids. The force field parameters of retinal were adapted from recent literatures. 8,9 The TIP3P water model was used. The energy of the system was first minimized with very strong position restraints to heavy atoms of the protein (including retinal and two palmitoyl chains bound to rhodopsin). The position of the phosphorus atom and two dihedrals of lipids were also restrained strongly. Then, the system was under a series of equilibration with position and dihedral restraints were gradually released. The early stage of equilibration was performed in the NVE ensemble, then all the subsequent simulations were switched to the NPT ensemble. The system was coupled to a Berendsen thermostat with τ t =1.0 ps to keep the temperature constant at 303.15 K, and a semiisotropic Berendsen barostat with τ p =5.0 ps to keep the pressure constant at 1 bar. 10 An integration time step of 2 fs was used with constraints to bonds involving hydrogen atoms using the LINCS algorithm. 11 Lennard-Jones interactions were calculated using S2