Lipid nanodiscs (POPC or EPL) were prepared as previously described . Lipids were re-solubilized with MSP buffer (20 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, pH 7.4) containing 200 mM and 500 mM Na-cholate for POPC and EPL lipids respectively. MSP1E3D1 was added to the resuspended lipids at a 1:85 and 1:60 MSP:lipid molar ratio for POPC and EPL lipids respectively. Nanodisc mixtures with lipids, Na-cholate, and MSP were incubated at 4 °C for 30 min. BioBeads SM-2 (Bio-Rad) were added (~0.5 g of beads per 1 mL volume) to remove Na-cholate and drive nanodisc self-assembly. The MSP:lipid:cholate reconstitution was incubated with beads for at least 8 h with at least three bead changes. Beads were removed by filtration and generated nanodiscs were then purified using a Superdex 200 10/300 Increase GL column (GE Healthcare) in MSP buffer (Figure ). Purity and size were assessed by SDS-PAGE and dynamic light scattering (DLS) using a Particle Size Analyzer LiteSizer 500 (Anton Parr).