R 2 2 carbon film

Venetin-1 at a concentration of 1 mg mL⁻¹ was ultrasonicated for 10 min at 40 °C in an ultrasonic chamber (Pol-Sonic, Poland). Then, the sample was vitrificated in a water solution on the TEM grid covered with Quantifoil R 2/2 carbon film (Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Before observations, the grids were activated with oxygen plasma for 15 s in a Femto plasma cleaner (Diener Electronic, Germany). Next, 3 μL of the Venetin-1 suspension was transferred onto the grid, blotted with filter paper and immersed in liquid ethane for instant freezing by Vitrobot Mark IV (FEI Company, USA). Before observations, the samples were stored in liquid nitrogen. To transfer the specimens to the TEM microscope, they were loaded into the Gatan 626 Cryo-TEM holder (Gatan Inc., USA)^{59 (link)}. The samples were observed using a Tecnai F20 X TWIN microscope (FEI Company, USA) with 200-kV acceleration voltage of the emission gun. An Eagle 4k HS camera (FEI Company, USA) was used to record the images.

The CPV14 crystal slurry was diluted 1 in 12 into a solution of ethyl­ene glycol to give a final ethyl­ene glycol concentration of 50%(v/v). Ethyl­ene glycol was added to allow for finer control over the subsequent blotting process and to ensure cryoprotection of the crystals.
Crystals were cryocooled on electron-microscopy grids in preparation for further analysis. Cu 200 mesh grids coated with Quantifoil R 2/2 carbon film (Quantifoil) or Cu 400 mesh H7 finder grids with holey carbon (AgarScientific) were glow discharged before application of the sample. A 2 µl aliquot of 50%(v/v) ethyl­ene glycol was applied to the Cu side of the grid, followed by application of 2 µl of the diluted crystal slurry onto the carbon film. The grid was then blotted for 3.0–5.5 s from the Cu side of the grid using a Leica EM GP (20°C, humidity 90%). Blotted grids were then plunge frozen in liquid ethane. Grids were stored under liquid nitro­gen until required.