(a) The ThermoFluor (Alexandrov et al., 2008) and monodispersity validation of different constructs of DRD4. The melting temperature (Tm) of WT is approx. 43°C (i, blue curve, solid line, Boltzmann sigmoid fitting, dashed line, experimental data), and the F3.41W mutation construct elevated Tm ~5°C (panel i, red curve). Tm of the BRIL-fusion protein reached above 50°C (panel ii, black curve), and when it was incubated with DRD4 ligands Tm was raised above 70°C (ii, colored curves). The size exclusion chromatography assay with nanofilm SEC-250 (Sepax Technologies) shows that the monodispersity of the fusion protein (panel iii) was improved differently with different ligands. 0: no ligands during purification; 1: thioridazine (Seeman and Lee, 1975) (Sigma-Aldrich); 2: ABT-724 (Brioni et al., 2004) (Tocris); 3: (-)quinpirole (Millan et al., 2002) (Tocris); 4: PD-168568 (Belliotti et al., 1998) (Tocris); 5: Mesoridazine (Choi et al., 2004) (Sigma-Aldrich); 6: Fananserin (Truffinet et al., 1999) (Sigma-Aldrich); 7: spiperone (Hidaka et al., 1995) (Sigma-Aldrich); 8: L745870; and 9: L750667 (Schetz et al., 2000) (Tocris). (b) The DRD4-L745870 complex shows a single peak in Superdex200 (R.V., retention volume). The Tm of the complex was determined as ~72°C. (c) Initial crystal images under different conditions (panels i–iv: visual light image) and optimized crystals (panel v: visual light image and polarized light image) used for data collection.