Structural Determination of β2AR-T4L and Nb80 Complex

Preparation of β₂AR-T4L and Nb80 are described in Supplementary Methods. BI-167107 bound β₂AR-T4L and Nb80 preincubated in 1:1.2 molar ratio were mixed in monoolein containing 10% cholesterol in 1:1.5 protein to lipid ratio (w/w). Initial crystallization leads were identified and optimized in 24-well glass sandwich plates using 50 nL protein:lipid drops overlaid with 0.8 μl precipitant solution in each well and sealed with a glass cover slip. Crystals for data collection were grown at 20° C in hanging-drop format using 0.8 μl reservoir solution (36 to 44% PEG 400, 100 mM Tris pH 8.0, 4 % DMSO, 1 % 1,2,3-heptanetriol) diluted 2 to 4-fold in water. Crystals grew to full size, typically 40 x 5 x 5 μm³, within 7 to 10 days. Crystals were flash frozen and stored in liquid nitrogen with reservoir solution as cryoprotectant. Diffraction data collection and processing, and structure solution and refinement are described in Supplementary Methods.