Topic: Cooling fast and slow: Recovering equilibrium statistics from vitrified cryo-EM samples
Abstract: Cryogenic electron microscopy (cryo-EM) relies on the rapid cooling of biological samples to form vitreous ice, preserving molecular structures for high-resolution imaging. Real-world plunge-vitrification occurs at a rate that may potentially overestimate lower-energy structures and obscure alternative conformations1. This non-equilibrium perturbation of the conformational ensemble has significant implications for interpreting cryo-EM data, particularly in time-resolved studies, structural heterogeneity analysis, and the direct recovery of ensembles from cryo-EM2. Here, we explore plunge-vitrification of full conformational ensembles and study its effect upon energy landscapes at cooling rates at the known limits for vitreous ice formation3. We first used temperature replica-exchange molecular dynamics simulations to obtain a landscape of the known fast-folding protein Trp-Cage from 230 K to 510 K. We used the landscape at 277 K to seed thousands of unbiased simulations across the folding landscape and obtained a Markov state model to precisely quantify equilibrium kinetic behavior. We then used thousands of rapid quench molecular dynamics simulations at different realistic cooling rates to uncover the relationship between molecular kinetics and their non-equilibrium perturbation under vitrification. Finally, we derived strict thermodynamic constraints on the possible distributions of quenched populations, which in turn enabled us to infer the original equilibrium ensemble from quenched ensembles alone. This thermodynamic approach to distribution inference could enable direct reconstruction of equilibrium free-energy landscapes of proteins from cryo-EM populations.