To isolate total membranes, ~7 g of BL21 Star (DE3)pLysS cells expressing BtuCD was suspended in 130 mL of ice-cold 50 mM MOPS buffer (pH 7.5) containing 60 mM NaCl containing, 10 µg/mL DNase Ι and one protease inhibitor cocktail tablet (Roche, 4693159001). Cells were lysed by sonication and the lysate was centrifuged at 15,000xg for 20 min at 4°C. The total membrane fraction (~ 1.3 g) were collected by ultracentrifugation at 200,000xg for 1.5 h.