Mature 80S ribosomes were isolated using two different protocols from the HEK Expi293F cells as described elsewhere with modifications (Khatter et al. 2014 (link), 2015 (link); Bhaskar et al. 2020 (link)). The ribosomal pellet obtained in the final step was then resuspended in resuspension buffer (50 mM HEPES 7.6, 80 mM Potassium acetate, 5 mM Magnesium chloride, and 1 mM DTT) to obtain a final concentration of ribosomes at 0.5–0.7 mg/mL. A total of 4 µL of the sample was applied on the Quantifoil R2/2 Cu 300 mesh grids (Quantifoil, Micro Tools GmbH). The grids were blotted for 3 sec at 4°C and 80% humidity and subsequently plunge frozen in liquid ethane using Leica EM GP plunge freezer (Leica Microsystems). Alternatively, 4 µL of the sample was applied on the Quantifoil R2/2 Cu 300 mesh grids coated with 3 nm continuous carbon (Quantifoil, Micro Tools GmbH). The grids were blotted for 3 sec at 4°C and 80% humidity and subsequently plunge frozen in liquid ethane using Vitrobot (Thermo Fisher Scientific).