Structural Determination of GABA(B) Receptor-G(i1) Complex

The GB1 and GB2 plasmids mixed with PEI 25 K at a 3:0.5:0.5 ratio of PEI to GB1 and GB2 plasmid (w/w) were added to HEK293F cells when the density reached about 2.8 million per ml. Seventeen hours after infection, sodium butyrate was added at a final concentration of 10 mM and the cells were grown for another 3 days at 30 °C before being collected^{11 (link)}. The collected cells were solubilized for 3 h at 4 °C in a buffer containing 0.5% (w/v) lauryl maltose neopentyl glycol (Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (Anatrace). After centrifugation at 30,000g for 30 min, the GABA_B was purified by Ni-NTA column and M1 anti-Flag affinity resin. The GABA_B was further concentrated and mixed with a 1.3 molar excess of G_i1–scFv16 complex in the presence of 100 μM baclofen and 50 μM BHFF. The sample was incubated at 25 °C for 1 h, followed by the addition of 0.2 U ml⁻¹ apyrase for an additional 1.5-h incubation at 24 °C^{36 (link)}. Finally, the sample was purified using a Superose 6 Increase column (GE Healthcare) to acquire a homogeneous GABA_B receptor–G_i1 complex. The entire purification procedure was accomplished in 12 h, followed by immediate verification to acquire a stable and fresh sample for structural determination.