Frozen in time: analyzing molecular dynamics with time-resolved cryo-EM

Molecular machines, such as polymerases, ribosomes or proteasomes, fulfil complex tasks requiring the thermal energy of their environment. They achieve this by restricting random motion along a path of possible conformational changes. These changes are often directed through engagement with different cofactors, which can best be compared to a Brownian ratchet. Many molecular machines undergo three major steps throughout their functional cycles, including initialisation, repetitive processing, and termination. Several of these major states have been elucidated by cryogenic electron microscopy (cryoEM). However, the individual steps for these machines are unique and multi-step processes, and their coordination in time is still elusive. To measure these short-lived intermediate events by cryoEM, the total reaction time needs to be shortened to enrich for respective pre-equilibrium states. This approach is termed time-resolved cryoEM (trEM). In this review, we sum up the methodological development of trEM and its application to a range of biological questions.