Figure 2.

Time-resolved cryo-EM allows visualizations of intermediate states of molecules in action

A spectacular time-resolved cryo-EM (trCryo-EM) study (Loveland et al., 2020) elucidating 33 states involved in tRNA proofreading process of the elongation step of protein synthesis is presented here. A schematic representation of the trCryo-EM technique is shown in the middle where different components (represented in red and yellow colored test tubes) are allowed to interact (mixing of components is shown in red and yellow color through a zigzag channel) and subsequently sprayed on the grids at different time points. Grids are plunged frozen and imaged by high-resolution cryo-TEM. The crests and troughs of the zigzag line are the time points where at each point the sample can be deposited onto the grid for image acquisition. Five representative structures of ribosomes (shown in different shades of blue) in the complex with EF-Tu are shown as for example (emd-21621, emd-21623, emd-21624, emd-21626, and emd-21629) revealing EF-Tu (shown in red) conformational changes during the process of tRNA delivery and subsequent release of EF-Tu upon GTP hydrolysis. The sequential intermediate structures trace the conformational changes from ribosome binding of EF-Tu in GTP-bound form (state I; GTP shown in green) to a state where EF-Tu is on the verge of leaving the ribosome upon GTP hydrolysis (state V).