The CHARMM-GUI web server was then used to create hydrated, membrane-bound MCR-1 systems for MD simulations . The protein was oriented in the membrane using the PPM server option on the protein chain. A heterogeneous lipid bilayer was built in a rectangular box with the Z dimension determined by the thickness of the water shell (20 Å) and the X and Y dimensions by the numbers of lipid components, keeping the two values equal to one another. The bilayer was composed of a mixture of palmitoyloleoyl phosphoethanolamine (POPE) and palmitoyloleoyl phosphatidylglycerol (POPG) lipids in an approximate 80:20 ratio. The upper leaflet was comprised of 302 POPE and 75 POPG lipids, and the lower leaflet of 320 POPE and 80 POPG lipids, to account for the reduced intracellular cross-sectional area of the protein relative to the extracellular area. This produced a final system size of c. 156 x 156 x 124 Å. The membrane was built using a replacement method, which involved one POPE residue being modelled into the active site. CaCl2 ions were included using a distance ion placing method to neutralize the system, with a final concentration of 0.1 M. Input files were generated by the web server for AMBER using NPT ensemble at 300 K, using ff14SB for protein, lipid17 for lipids, and TIP3P for water after membrane building . The bound zinc ion(s) were parameterised using the standard 12-6 Lennard-Jones (LJ) nonbonded model . During the membrane building step, a POPE phospholipid was modelled into the active site; this was deleted from the system in models without bound phospholipid. The various systems were minimized using the AMBER engine pmemd.MPI for 500 steps using a non-bonded cut-off of 9.0 Å, with harmonic positional restraints on the protein (weight 10.0), and on the membrane (weight 2.5). NPT equilibration was performed at 300 K (except where stated) using the Berendsen barostat and Langevin dynamics with γ = 1.0 ps -1 , a nonbonded cut-off of 9.0 Å, and applying SHAKE 31 restraints. The equilibration was performed over six steps with parameters specified in Table . For each system tested three production runs, each of 500 ns, were then carried out.