1.2 1 3 grid

Purified hIP₃R-3 in the SEC buffer containing 1 mM EDTA was supplemented with 500 µM IP₃ (from 10 mM stock in water), 0.1 mM CaCl₂, and 5 mM ATP (from 100 mM stock, pH 7.2). 2.0 μL of the protein sample was applied to 300 mesh Cu Quantifoil 1.2/1.3 grids (Quantifoil Microtools) that were glow discharged for 20 s at 25 mA. The grids were blotted for 7 s at force 10 using single-layer Whatman ashless filter papers (Cat. #: 1442-055, GE Healthcare) and were plunged into liquid ethane using an FEI MarkIV Vitrobot at 8 °C and 100% humidity. The filter papers were not pre-treated with Ca²⁺ chelators or any other chemicals. Four grids prepared using the same sample were imaged using a 300 kV FEI Krios G3i microscope equipped with a Gatan K3 direct electron camera in four different data collection sessions at Case Western Reserve University. Movies containing 40–50 frames were collected at a magnification of ×105,000 in super-resolution mode with a physical pixel size of 0.828 Å/pixel and defocus values at a range of −0.8 to −1.6 µm using the automated imaging software SerialEM^{47 (link)} and EPU (ThermoFisher Scientific).

Purified tick-borne encephalitis virus was deposited onto pre-clipped Quantifoil 1.2/1.3 grids with a 2 nm continuous carbon layer using 150 µm diameter pins. Bacteriophage FJy-3 was purified from its host, Flavobacterium sp. B169, in 20 mM potassium phosphate pH 7.2. The sample was deposited onto pre-clipped Quantifoil holey carbon R1.2/1.3 Cu 300 mesh grids in the VitroJet. Prior to sample deposition, the grids were plasma-cleaned in the VitroJet for 75 s. For pin printing, a circular spiral pattern was used with a speed of 1 mm s⁻¹, a spacing of 120 µm and a standoff distance of 10 µm.
Cryo-EM grid screening and data collection were performed at the cryo-EM facility in Helsinki, Finland using a Thermo Fisher Scientific Talos Arctica operating at 200 kV and equipped with a Falcon 3 direct electron detector operating in linear mode. Images were collected at 57 000× magnification with an image size of 4096 × 4096 at a sampling rate of 0.26 nm per pixel.

5-HT_3AR protein (~2.5 mg/ml) was filtered and incubated with 100 μM drugs (Alosetron, Ondansetron, and Palonosetron) for 1 hr. Fluorinated Fos-choline-8 (Anatrace) was added to the protein sample to a final concentration of 3 mM. The protein was then blotted onto Cu 300 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools) two times with 3.5 μl sample each time, and the grids were plunge frozen immediately into liquid ethane using a Vitrobot (FEI). The grids were imaged using a 300 kV FEI Titan Krios G3i microscope equipped with a Gatan K3 direct electron detector camera. Movies containing ~50 frames were collected at 105,000 × magnification (set on microscope) in super-resolution mode with a physical pixel size of 0.848 Å/pixel, dose per frame 1 e^-/Å (Gilmore et al., 2018 (link)). 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 SerialEM (Mastronarde, 2005 (link)).

For all samples, fluorinated Fos-Choline-8 was added to the concentrated protein to a final concentration of 3 mM immediately prior to vitrification. Samples were double blotted on 200 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools) with 3.5 µL per blot and plunge frozen in liquid ethane using a Vitrobot (Thermo Fisher Scientific). Grids containing TRPV5 and diC₈ PI(4,5)P₂ in detergent, as well as grids with CaM-bound TRPV5 were imaged with a 300 kV Titan Krios microscope equipped with a Gatan K2 Summit direct detector camera. Super resolution movies (50 frames) were captured for 10 s each with one frame collected every 0.2 s. The resultant pixel size and dose rate were 0.55 Å/pix and ~8 electrons/pix/s, respectively. Images were collected in a defocus range between 1.0 and 2.5 µm under focus in an automated fashion using Leginon software^{33 (link)}.
Grids containing TRPV5 and diC₈ PI(4,5)P₂ in nanodiscs were also imaged with a 300 kV Titan Krios microscope equipped with a Gatan K2 Summit direct detector camera. Super resolution movies (40 frames) were captured with an 8 s exposure time in super resolution mode resulting in a pixel size and dose rate of 0.532 Å/pix and ~6 electrons/pix/sec, respectively. Images were collected in a defocus range between 1.25 and 2.5 µm under focus.