CryoEM grid preparation

Samples for grid preparation were prepared as follows. Purified BAM-LL or BAM-P5L in 50 mM Tris–HCl pH 8.0, 150 mM NaCl and 0.05% (w/v) DDM were diluted to 3.3 or 2.3 mg/mL, respectively. For the BAM–Fab1 complex, purified WT BAM was mixed with a 2-fold molar excess of Fab1 and run on a Superdex 200 10/300 column in TBS pH 8.0, 0.05% (w/v) DDM to isolate a stoichiometric complex from excess free Fab1. Fractions corresponding to the complex were concentrated to 4.8 µM using Vivaspin 500 concentrators MWCO 30k (Sartorius). To assemble the Fab1-bound BAM-LL complex, stock solutions of purified BAM-LL and Fab1 were first diluted to 5.9 μM in 20 mM Tris–HCl pH 8.0, 150 mM NaCl and 0.05% (w/v) DDM and mixed in a 1:1 molar ratio, before dilution in detergent-free buffer to a total protein concentration of 0.9 mg/mL and a total DDM concentration of 0.03% (w/v). The detergent concentration was lowered to combat a tendency for very thin ice on the resulting grids.

CryoEM grids were prepared as follows. For the BAM–Fab1 complex, 4 µL protein was applied to gold UltrAUfoil R2/2 200 mesh grids, previously glow discharged for 60 s at 20 mA in a GlowQube Plus (Electron Microscopy Sciences) in the presence of amylamine vapour. For BAM-LL, BAM-P5L and BAM-LL in complex with Fab1, 3 μL of sample was applied to copper QUANTIFOIL R1.2/1.3 300 mesh, copper QUANTIFOIL R0.6/1 400 mesh and gold UltrAUfoil R1.2/1.3 300 mesh grids (Electron Microscopy Sciences), respectively, that had been glow discharged for 30 s at 60 mA in a GlowQube Plus (Electron Microscopy Sciences). Grids were blotted for 6 s with Whatman #1 filter paper at 4 °C and 80–100% relative humidity, before plunge freezing in liquid ethane using a Vitrobot Mark IV (ThermoFisher).