AcrB was purified in DDM as described previously . After purification, AcrB was inserted into nanodiscs according to the previously established protocols . Briefly, AcrB in 0.03% (w/v) DDM detergent was mixed with POPC and MSP solution at a final 40:1:0.5 lipid:MSP1E3D1:AcrB molar ratio in MSP buffer with final concentration of 0.0116% (w/v) DDM and 16 mM Na-cholate. DDM was removed by the addition of SM2 Bio-beads (Bio-Rad) into the mixture and left in an orbital shaker overnight at 4 °C. AcrB nanodiscs were purified using a Superdex 200 Increase 10/300 (GE Healthcare) in AcrB sample buffer (50 mM sodium phosphate, 150 mM NaCl, 10% glycerol, pH 7.4) (Figure ).