Structural dynamics: review of time-resolved cryo-EM

Abstract

The structural determination of biological macromolecules has been transformative for understanding biochemical mechanisms and developing therapeutics. However, the ultimate goal of characterizing how structural dynamics underpin biochemical processes has been difficult. This is largely due to significant technical challenges that hinder data collection and analysis on the native timescales of macromolecular dynamics. Single-particle cryo-EM provides a powerful platform to approach this challenge, since samples can be frozen faster than the single-turnover timescales of most biochemical reactions. In order to enable time-resolved analysis, significant innovations in the handling and preparation of cryo-EM samples have been implemented, bringing us closer to the goal of the direct observation of protein dynamics in the milliseconds to seconds range. Here, the current state of time-resolved cryo-EM is reviewed and the most promising future research directions are discussed.

Biological function investigated by time-resolved structure determination

“…Recent advances in single particle cryo-EM allow structures to be determined with near atomic resolution that rivals that of crystallography. However, time-resolved methods need to be implemented that are fast enough to cover macromolecular reactions in a meaningful way ( Maeots and Enchev, 2022 ).…”

Fast Viral Dynamics Revealed by Microsecond Time-Resolved Cryo-EM

“…The pH induced contraction of a another icosahedral virus, Nudaurelia Capensis ω, was observed to be complete after 10 ms, but is thought to be significantly faster (7). This suggests that the capsid motions of CCMV would be too fast to be captured by traditional timeresolved cryo-EM, which affords only millisecond time resolution (8). Ultrafast x-ray crystallography (9, 10), while sufficiently fast, is likely not suitable either to study these dynamics, since the crystal environment would hinder the large-amplitude motions involved (10,11).…”

Exploring the Structural Variability of Dynamic Biological Complexes by Single-Particle Cryo-Electron Microscopy

“…Their functional variability often involves structural transitions with transient intermediates representing only a low percentage of the population at equilibrium. Despite their importance to understanding the mechanism of action of molecular machines, these short-lived states have remained elusive to traditional structure determination methods because their lifetime is several orders of magnitude shorter than the time required for sample preparation [ 69 ]. Time-resolved cryo-EM (trEM) is a rapidly developing branch of cryo-EM used to study transient structural intermediates by stopping a biochemical reaction at fixed time points via vitrification.…”

“…Recently, several “fast” mixing methods have been developed, including on-grid mixing, also known as the spraying-mixing method [ 73 , 74 , 75 ], microfluidic mixing-spraying [ 32 , 76 ], and light pulse delivery [ 77 , 78 ]. On-grid mixing, developed by Berriman and Unwin [ 73 ] and reviewed in [ 69 ], requires the direct application of the first reactant onto a grid, thinning of the solution via blotting, and spraying aerosol droplets of the second reactant onto the grid just before vitrification ( Figure 2 A). This approach has been used to successfully determine cryo-EM structures of the open channel form of acetylcholine receptor [ 67 ], the conformational changes of myosin attached to actin during the ATPase cycle [ 74 ], and vesicle formation [ 79 ].…”

“…Microfluidic mixing-spraying has been applied to study multiple protein–protein interactions, including ribosome subunit association [ 65 , 66 ] and intermediates of translation initiation [ 88 ]. Notwithstanding, to achieve desired flow rates (~6 μL/s) and sufficient particle distribution on the grid [ 69 ], this approach requires larger volumes of sample at higher concentrations compared to those needed in traditional blotting methods. These factors limit the applicability of microfluidic mixing-spraying for macromolecules that are difficult to purify.…”