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.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.