Effects of cryo-EM cooling on structural ensembles of biomolecules and their solvation shell.

Abstract

The recent revolution in cryo electron microscopy (cryo-EM) allows the determination of structures of macromolecular complexes at atomic resolution. Cryo-EM also provides information on structural heterogeneity and ensembles of macromolecules. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to liquid nitrogen temperatures. The rapid cooling prevents ice-crystal formation and preserves some information of the room-temperature ensemble. However, to what extent the ensemble of the macromolecues and of the solvation shell is perturbed by the cooling is currently unknown. To quantify the cooling effects, we first estimated the temperature drop rate by solving the heat equation which suggested rapid cooling within microseconds. Then, we started all-atom explicit-solvent molecular dynamics (MD) simulations of the ribosome from 41 snapshots taken from a room-temperature ensemble with linearly decreasing temperatures at 11 different cooling rates (simulation lengths 0.1-128 ns). In the simulations, we observed that cooling markedly decreased the structural heterogeneity of the cooled ensemble. To investigate if and how this effect depends on the cooling rate, we used Bayesian statistics to test three thermodynamic and kinetic models of the cooling process. A kinetic two-state model improves the prediction of the decrease in heterogeneity compared to the cooling-rate independent thermodynamic model suggesting that kinetic effects contribute markedly. The combination of the estimated temperature drop rate with the kinetic model suggests that small barriers between states (<10 kJ/mol) are overcome during cooling and do not contribute to the heterogeneity of the structural ensemble obtained by cryo-EM. In contrast, conformational states separated by larger barriers are expected to be trapped during plunge-freezing. The obtained parameters for the kinetic model will allow one to quantify the heterogeneity of biologically relevant room-temperature ensembles from cryo-EM structures.