Serotonin-Induced 5-HT3A Receptor Structure

Functional characterization shows that serotonin-induced 5-HT_3AR currents saturate at 30 μM and beyond^{11 (link),33 (link),34 (link)}. Therefore, the 5-HT_3AR protein (~2.5 mg/mL) was filtered and first incubated with 100 μM serotonin for 30 min. After which, 3 mM Fluorinated Fos-choline 8 (Anatrace) was added and the sample was incubated until blotting^{35 (link)}. The sample was blotted twice with 3.5 μL sample each time onto Cu 300 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools), and immediately after the second blot, the grid was plunge frozen into liquid ethane using a Vitrobot (FEI). The grids were imaged using a 300 kV FEI Titan Krios microscope equipped with a Gatan K2-Summit direct detector camera. Movies containing 40-frames were collected at 130,000x magnification (set on microscope) in super-resolution mode with a physical pixel size of 0.532 Å/pix, dose rate of 3.754 electrons/pix/s, and a total exposure time of 12 seconds. Defocus values of the images ranged from −1.0 to −2.5 µm (input range setting for data collection) as per the automated imaging software Latitute S (Gatan Co.).