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

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.

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.