Purified TRPC5 protein sample (3.5 μl) in PMAL-C8 at 7.0 mg/ml was applied to glow-discharged Quantifoil R1.2/1.3 holey carbon 400-mesh copper grids (Quantifoil). Grids were blotted for 7 s at 100% humidity and flash frozen by liquid nitrogen–cooled liquid ethane using a FEI Vitrobot Mark I (FEI). The grid was then loaded onto FEI TF30 Polara electron microscope operated at 300-kV accelerating voltage. Image stacks were recorded on a Gatan K2 Summit direct detector (Gatan) set in super-resolution counting mode using SerialEM (35), with a defocus range between 1.5 and 3.0 μm. The electron dose was set to 8 e−/physical pixel per s, and the subframe time was set to 0.2 s. A total exposure time of 8 s resulted in 40 subframes per image stack. The total electron dose was 42.3 e−/Å2 (~1.1 e−/Å2 per subframe).

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