bioRxiv2026
Single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution structure determination of diverse biomolecules. Because the high vacuum required for electron microscopy prevents the imaging of liquid-phase samples, cryo-EM samples are prepared by plunging the sample into a cryogen, rapidly cooling the sample and suspending the ensemble of biomolecules in a matrix of water glass. However, the effects of this vitrification on the biomolecular ensemble are unknown, complicating efforts to use cryo-EM to derive conformational ensembles of biomolecules. To study these effects, we carried out extensive molecular dynamics simulations (over 50 milliseconds) of the Trp-cage miniprotein at equilibrium and undergoing rapid cooling. We simulated seven cooling rates spanning three orders of magnitude, with the slowest coolings matching experimental rates. By inspecting molecular mobility and density-temperature equations of state for water with and without protein, we found that water vitrification is unaltered by the protein. To track protein conformation changes, and to relate them to conformational kinetics, we made a Markov State Model (MSM) of Trp-cage from 5.4 milliseconds of equilibrium sampling at 277 K. We observed that MSM states with a characteristic time longer than the duration of the non-equilibrium cooling, tend to be more robust to artefacts induced by such cooling. Critically, although we observe that some states vanish in the equilibrium ensemble at 230 K, none do in our nonequilibrium cooled ensembles. However, to account for perturbations induced by nonequilibrium cooling for more labile states, we developed a thermodynamic inference framework for recovering equilibrium populations from the measured vitrified ensembles. These results indicate that cryo-EM has the capacity to be a reliable and accurate biophysical technique for the study of biomolecular ensembles. SignificanceCryogenic electron microscopy images biomolecules trapped in vitreous ice. To vitrify the sample, it must be cooled over the course of 22 microseconds. However, the degree to which this cooling causes the ensemble of the molecules to be perturbed from equilibrium is unknown. Here we present extensive molecular dynamics simulations to quantify the equilibrium dynamics of the Trp-cage miniprotein and the effects of cooling on its conformational ensemble. By simulating cooling at seven different rates, including the slowest experimental rates that still result in vitrification, we connect the kinetic properties of a proteins conformational state to the change in state population from cooling. We show that cooling-induced population shifts are small but observable. We further introduce a thermodynamic-inference method to recover equilibrium populations from the cooled ensembles.