All atom molecular dynamics simulations were performed with AMBER starting with the crystal structure of SERCA1A in the E1.Mg2+ state from Oryctolagus cuniculus (PDB: 3W5A) . In preparation for simulation, all ligands and ions were removed from the 3W5A structure, and ATP or dATP were manually docked in the nucleotide binding pocket based on the ATP position in 7BT2, which has the nucleotide bound . Protein protonation state was determined based on PropKa at a pH of 7 . SERCA was oriented for placement into a lipid bilayer according to the Orientations of Proteins in Membranes (OPM) database . CHARMM-GUI was used to build the membrane and solvate the system in a 12.5 by 12.5 nm lipid bilayer composed of POPC (51% upper leaflet, 66% lower leaflet), POPE (43% upper leaflet, 17% lower leaflet), and POPS (6% upper leaflet, 17% lower leaflet) experimentally determined membrane composition in the cardiac SR . The water box padding was set to 22.5 nm and 150 mM K + Cl -ions were added according to Monte Carlo placement. The dATP structure was constructed by removing the extra hydroxyl from the ATP structure in 7BT2. Both ligands were parameterized using antechamber to generate AMBER GAFF2 force field parameters . The proteins and lipids were parameterized according to AMBERFF19FSB and Lipid17 with OPC water molecules . All molecular dynamics simulations were carried out on the Triton Shared Computing Cluster (TSCC). The production runs for each SERCA condition (apo, ATP, and dATP) were each 312 ns. Prior to the production simulation, minimization was carried out over 5000 steps of steepest descent minimization with 10 kcal mol -1 Å-2 positional restraints on all protein atoms and 2.5 kcal mol -1 Å-2 positional restraints on all lipid atoms, with NMR restraints. A six step equilibration approach was carried out for 1.875 ns total. During the first two steps, an NVT Langevin equilibration scheme was applied where 10 kcal mol -1 Å-2 positional restraints were present on all protein atoms for the first step and 5 kcal mol -1 Å-2 positional restraints were present on all protein atoms for the second step. Lipids were restrained using 2.5 kcal mol -1 Å-2 positional restraints with NMR restraints were present on all lipid atoms for both steps. The next four stages used an NPT ensemble for 125 ps, 500 ps, 500 ps, and 500 ps, respectively, with the system set to a pressure of 1.0 bar. All atom protein restraints set decreased through these stages and were specified at 2.5, 1.0, 0.5, and 0.1 kcal mol -1 Å-2 respectively. Similarly, positional and restraints on all lipid atoms were 1.0, 0.5, 0.1, and 0 kcal mol -1 Å-2 for each step, respectively, with NMR restraints. Triplicate production MD simulations were run at 303.15 K using the PMEMD (Particle Mesh Ewald Molecular Dynamics) method with a 9 Å nonbonded cutoff, and 2 fs timestep. The first triplicate was ran for 312.25 ns coordinates saved every 5 ps leading to 62,450 frames, while the second and third triplicate were each ran for 300 ns with 60,000 frames per simulation for replicates 2 and 3. The mutual information algorithm was applied to the first triplicate with all 62,450 frames, as well as just the first 60,000 frame with no significant change. Therefore we selected to use all available data and include the full trajectory. Images were generated using VMD .