Crystallization of the gel-filtrated rhodopsin/mini-Go complex was set up using the vapor diffusion method. Crystallization drops were dispensed using a mosquito crystallization robot by mixing 200 nl of protein and 200 nl of crystallization buffer containing 0.1 M MES (pH 5.5) and 10 to 20% polyethylene glycol 4000 (PEG 4000) in an MRC 2-well crystallization plate (Swissci) at 4°C. Sword- or rod-shaped crystals appeared in 1 to 3 days, and crystals wider than 10 μm mainly grew from drops using the crystallization buffer containing 13 to 17% PEG 4000. Before crystals were harvested on crystal loops (MiTeGen), crystals were cryoprotected by adding 400 nl of mother liquor supplemented with 10% glycerol or 10% trehalose to the crystallization drop 1 day before harvesting. Harvested crystals were flash-frozen in liquid nitrogen (fig. S1B). Frozen crystals were evaluated at the PXI beamline at the Swiss Light Source (SLS) using raster scanning to identify the best diffracting locations on a frozen crystal. Diffraction datasets were collected by exposing crystals to a 10- or 20-μm-sized beam with 0.05° oscillation angle per frame.

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