Ultrasonic chamber

The AAF solution with a protein concentration of 1 mg mL⁻¹ was placed in an ultrasonic chamber (Pol-Sonic, Poland) for 10 min at 40°. Next, the preparation of the AAF specimen consisted in vitrification of an aqueous suspension on the TEM grid with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using the Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). The samples of AAF were vitrified by applying a droplet (3 μL) of the suspension to the grid, blotting with filter paper, and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, Hillsboro, Oregon, USA). After preparation, the vitrified specimens were kept in liquid nitrogen until they were inserted into the Cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, USA) providing a sufficiently low temperature (− 178 °C) during the transfer of the samples to the microscope and during the TEM analyses^{25 (link)}. Cryogenic Transmission Electron Microscopy (Cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a field emission gun (FEG) operating at the acceleration voltage of 200 kV. Images were recorded with an Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA).

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 Venetin-1 solution with a protein concentration of 1 mg/mL was placed in an ultrasonic chamber (Pol-Sonic, Poland) for 10 min at 40 °C. The aqueous suspension was then vitrified on a TEM mesh with a punched carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). The mesh was previously activated for 15 s in oxygen plasma using a Femto device (Diener Electronic, Ebhausen, Germany). A 3-µL sample was applied to the mesh. Thereafter, blotting with filter paper and instant freezing in liquid ethane using a Vitrobot Mark IV blower (FEI Company, Hillsboro, Oregon, USA) were performed. Glazed samples were kept in liquid nitrogen until placing in a Cryo-TEM Gatan 626 holder (Gatan Inc., Pleasanton, USA). Cryogenic Transmission Electron Microscopy (cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a Field Emission Cannon (FEG) operating at an acceleration voltage of 200 kV. An Eagle 4 k HS camera (FEI Company, USA) was used for image recording and TIA software (FEI Company, USA) was used for processing.