Cryo-EM Imaging of Ribosome Complexes

3 μL aliquots of purified ribosome complexes at a concentration of 120 nM were applied onto Quantifoil R2/2 cryo-EM grids covered with continuous carbon (estimated to be 50 Å thick) at 4°C and 100% ambient humidity. After 30 s incubation, the grids were blotted for 3 s and vitrified in liquid ethane using a Vitrobot MKIII (FEI).
Automated data collection (EPU software, FEI) was conducted on a Titan Krios microscope equipped with a XFEG electron source using 300 kV acceleration voltage. For each 1.1 s exposure, 17 movie frames were recorded on a Falcon II direct electron detector (FEI) at a calibrated magnification of 104,478, resulting in a pixel size of 1.34 Å^{33 (link)}. A dose rate of ~30 electrons per Ų per second was used. Defocus values ranged from −1.1 to −5.9 μm for the UAA-eRF1^AAQ dataset, −0.7 to −4.1 μm for the UAG-eRF1^AAQ dataset, and −0.7 to −3.8 μm for the UGA-eRF1^AAQ dataset (Extended Data Table 1), as more images were collected closer to focus on the latter two datasets.