The CysLT1R-BRIL in complex with pranlukast was crystallized using the LCP method by mixing 40% of protein (50 mg ml−1) with 60% of lipid (monoolein and cholesterol, 9:1, w/w) using a syringe lipid mixer (14). After a clear LCP formed, the mixture was dispensed onto 96-well glass sandwich plates (Marienfeld) as 40-nl drops and overlaid with 800 nl of precipitant solutions using an NT8-LCP robot (FORMULATRIX). Crystals appeared after 2 to 10 days and reached their full size within 2 weeks using 100 mM sodium citrate (pH 6), 200 to 600 mM lithium nitrate, and 30 to 38% (v/v) polyethylene glycol 400 (PEG400) as a precipitant solution. Typical crystal size was 250 μm by 15 μm by 15 μm, and crystals had a twiggy shape (fig. S1D). Crystals were harvested directly from LCP using 100- to 200-μm MicroMount loops (MiTeGen) and flash frozen in liquid nitrogen. For the XFEL data collection, CysLT1R-BRIL purified with zafirlukast was crystallized in syringes as described (38), using 75 to 175 mM sodium phosphate, 24 to 34% (v/v) PEG400, 100 mM Hepes (рН 7), and 1 μM zafirlukast as a precipitant. Typical crystal size was 5 μm by 2 μm by 2 μm (fig. S1C).

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.