Mini-Go was prepared as described (6, 43). The N2C/M257Y/D282C mutant of bovine rhodopsin was expressed in human embryonic kidney (HEK) 293 GnTI cells as described (20, 44). Buffers for every purification step were cooled to 4°C before use, and the steps after the addition of 9-cis retinal were performed under dim red light before light activation of rhodopsin. HEK293 cells were homogenized in phosphate-buffered saline (PBS) buffer and then solubilized by supplementing dodecyl maltoside (DDM) (Sol-Grade, Anatrace) to 1.25% (w/v). After gentle mixing for 1 hour, the cell lysate was centrifuged at 200,000g for 1 hour to remove unsolubilized residual. The rhodopsin apoprotein in the solubilisate was captured using immunoaffinity agarose resin coupled to 1D4 antibody. The resin was collected and washed with 10 column volume (CV) of PBS buffer containing 0.04% DDM, and then 2 CV of PBS buffer containing 0.04% DDM and 50 mM 9-cis retinal (Sigma-Aldrich) or 11-cis retinal (National Institutes of Health and Hoffmann–La Roche) was mixed with the resin overnight in the dark. The resin was collected and washed sequentially with 15 CV of PBS buffer containing 0.04% DDM; 10 CV of buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, and 0.12% (w/v) OGNG (Anatrace); and 5 CV of buffer containing 20 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM MgCl2, and 0.12% OGNG. Dark-state rhodopsin was eluted thrice with 1.5 CV of buffer containing 20 mM Hepes (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.12% OGNG, and 80 μM 1D4 peptide TETSQVAPA for more than 2 hours each time. Eluted protein was concentrated using an Amicon Ultra concentrator [molecular weight cutoff (MWCO), 50,000] to 5 to 10 mg/ml. The concentrated protein was subjected to deglycosylation using the endoglycosidase F1 (Endo F1) at 1:100 (w/w) ratio overnight at 4°C. The deglycosydated rhodopsin was mixed with mini-Go at the molar ratio of 1:1.2 in the presence of apyrase (25 mU/ml; New England BioLabs), incubated for 30 min, and then irradiated with light passed through a 495-nm long-pass filter, followed by another 30-min incubation in the dark. The rhodopsin/mini-Go mixture was concentrated using an Amicon Ultra concentrator (MWCO, 50,000) and subjected to size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare) using a buffer containing 20 mM Hepes, 0.4 mM MgCl2, and 0.12% OGNG. The eluted fractions were examined by SDS–polyacrylamide gel electrophoresis (PAGE) (fig. S1D), and those fractions containing pure rhodopsin/mini-Go complex were pooled and concentrated to 8 mg/ml for crystallization. The ultaviolet-visible spectrum of the final sample was measured to optically evaluate the isoform of retinal in rhodopsin, showing an OD280/OD380 (ratio of optical density between 280 and 380 nm) of 1.8 in rhodopsin/mini-Go in contrast to 1.6 in light-activated rhodopsin.

The detergent OGNG was selected from a screening experiment, where rhodopsin was stable in both the dark and light states and upon mini-Go binding. Deglycosylation was a critical step for obtaining crystals of the rhodopsin/mini-Go complex. Peptide N-glycosidase F and Endo F1 were tested, and only Endo F1 rendered a homogeneous deglycosylated product, which was confirmed by SDS-PAGE (fig. S1C).

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