Human β2AR fused to an amino-terminal T4 lysozyme23 (link) was expressed and purified as described above. Following purification by alprenolol sepharose, the receptor was washed extensively with 30 μM of the low affinity antagonist atenolol while bound to FLAG affinity resin to fully displace alprenolol, then washed and eluted in buffer devoid of ligand to produce a homogeneously unliganded preparation. The receptor was then incubated for 30 minutes at room temperature with a stoichiometric excess of ligand (HBI or BI167107). A 1.3-fold molar excess of Nb6B9 was then added, and the sample was dialyzed overnight into a buffer consisting of 100 mM sodium chloride, 20 mM HEPES pH 7.5, 0.01% lauryl maltose neopentyl glycol detergent, and 0.001% cholesteryl hemisuccinate. In each case, ligand was included in the dialysis buffer at 100 nM concentration or higher. The sample was then concentrated using a 50 kDa spin concentrator and purified over a Sephadex S200 size exclusion column in the same buffer as for dialysis, and the β2AR-Nb6B9-ligand ternary complex was isolated. In the case of adrenaline, the low affinity and chemical instability of the ligand precluded overnight dialysis, so 100 μM adrenaline was added to receptor for 30 minutes at room temperature, then a 1.3-fold molar excess of Nb6B9 added and the sample was incubated for 30 minutes at room temperature. Following incubation, the sample was concentrated and immediately purified by size exclusion as above.
Following purification, samples were concentrated to A280 = 55 using a 50 kDa concentrator to minimize the detergent concentration in the final sample, then aliquoted into thin-walled PCR tubes at 8 μL per aliquot. Aliquots were flash frozen in liquid nitrogen and stored at -80 °C for crystallization trials. For crystallization, samples were thawed and reconstituted into lipidic cubic phase with a 1:1 mass:mass ratio of lipid. The lipid stock consisted of a 10:1 mix by mass of 7.7 monoacylglycerol (generously provided by Martin Caffrey) with cholesterol (Sigma). Samples were reconstituted by the two syringe mixing method10 (link) and then dispensed into glass sandwich plates using a GryphonLCP robot (Art Robbins Instruments). In the case of the β2AR-adrenaline complex, 1 mM fresh adrenaline was mixed with receptor prior to reconstitution. Crystals were grown using 30 nL protein/lipid drops with 600 nL overlay solution, which consisted of 18 – 24 % PEG400, 100 mM MES pH 6.2 to pH 6.7, and 40 – 100 mM ammonium phosphate dibasic. Crystals grew in 1 – 3 days, and were harvested and frozen in liquid nitrogen for data collection.
Following purification, samples were concentrated to A280 = 55 using a 50 kDa concentrator to minimize the detergent concentration in the final sample, then aliquoted into thin-walled PCR tubes at 8 μL per aliquot. Aliquots were flash frozen in liquid nitrogen and stored at -80 °C for crystallization trials. For crystallization, samples were thawed and reconstituted into lipidic cubic phase with a 1:1 mass:mass ratio of lipid. The lipid stock consisted of a 10:1 mix by mass of 7.7 monoacylglycerol (generously provided by Martin Caffrey) with cholesterol (Sigma). Samples were reconstituted by the two syringe mixing method10 (link) and then dispensed into glass sandwich plates using a GryphonLCP robot (Art Robbins Instruments). In the case of the β2AR-adrenaline complex, 1 mM fresh adrenaline was mixed with receptor prior to reconstitution. Crystals were grown using 30 nL protein/lipid drops with 600 nL overlay solution, which consisted of 18 – 24 % PEG400, 100 mM MES pH 6.2 to pH 6.7, and 40 – 100 mM ammonium phosphate dibasic. Crystals grew in 1 – 3 days, and were harvested and frozen in liquid nitrogen for data collection.
