Monoolein cholesterol

Purified M1-T4L•tiotropium and M4-mT4L•tiotropium were crystallized using lipid cubic phase technology. Each receptor was reconstituted by mixing the protein solution into 10:1 (w/w) monoolein:cholesterol (Sigma) in 1:1.5 parts w/w protein:lipid ratio using the two-syringe method^{24 (link)}. For the M1 receptor, samples of 50 nL (20–40 nL for M4) were spotted onto 96-well glass plates and overlaid with 800 nL (600 nL for M4) of precipitant solution for each well using a Gryphon LCP (Art Robbins Instruments). Glass plates were then sealed using a glass cover film and incubated at 20 °C. Initial crystals for the M1 receptor formed after 24 hours in conditions containing 33% PEG 300, 100 mM sodium acetate, and 100 mM Bis-Tris Propane (pH 8.0). For the M4 receptor, initial crystals formed after 24 hours in conditions containing 25–40% PEG 300, 50–100 mM EDTA (pH 8.0), and 100 mM MES (pH 5.5–6.5). M1 and M4 crystals were harvested using mesh grid loops (MiTeGen) and stored in liquid nitrogen before use.

LCP crystallizations of β₂AR-T4L in complex with alprenolol and AS408 were performed using a LCP crystallization robot (Gryphon, Art Robbins Instruments). In brief, protein solution was mixed with 9:1 (w/w) monoolein:cholesterol (Sigma) with protein to lipid ratio of 2:3 (w/w) and reconstituted into LCP using two-syringe method⁴⁵ . Then, 96-well glass sandwich plates were filled with 30 nl LCP overlaid with 1 μl precipitant solution and incubated at 20 °C. The best crystals were grown in conditions containing 0.1 M Tris-HCl, pH 8.0, 30–40% PEG400, 300–400 mM sodium formate, 6% 1,4-butanediol, 1 mM alprenolol, 1 mM AS408 and 1% DMSO.