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

Abstract

Molecular machines, such as polymerases, ribosomes, or proteasomes, fulfill 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 initialization, repetitive processing, and termination. Several of these major states have been elucidated by cryogenic electron microscopy (cryo-EM). However, the individual steps for these machines are unique and multistep processes themselves, and their coordination in time is still elusive. To measure these short-lived intermediate events by cryo-EM, the total reaction time needs to be shortened to enrich for the respective pre-equilibrium states. This approach is termed time-resolved cryo-EM (trEM). In this review, we sum up the methodological development of trEM and its application to a range of biological questions.

Keywords: SPA; molecular machines; time-resolved cryo-EM.

Figure 1:. Four cartoons depicting a selection of molecular machines where time-resolved cryoEM may prove particularly useful in illuminating their function.

A: Translation by the RNA polymerase II B: RNA splicing by the spliceosome C: 26S Proteasomal degradation D: Eukaryotic ribosome translation

Figure 2:. An illustrated overview of the different time-resolved cryoEM approaches reviewed in this article.

A: Manually timed sample mixing . B: Gas-assisted sample atomization . C: Voltage-assisted sample spray . D: Microfluidic mixing in silicon chips with gas-assisted spray . E: Laser-induced rapid in situ melting and revitrification . F: Laser-induced photolysis of caged reactants . G: Inkjet-based sample droplet application .