Effects of cryo-EM cooling on structural ensembles

Structure determination by cryo electron microscopy (cryo-EM) provides information on structural heterogeneity and ensembles at atomic resolution. To obtain cryo-EM images of macromolecules, the samples are first rapidly cooled down to cryogenic temperatures. To what extent the structural ensemble is perturbed during cooling is currently unknown. Here, to quantify the effects of cooling, we combined continuum model calculations of the temperature drop, molecular dynamics simulations of a ribosome complex before and during cooling with kinetic models. Our results suggest that three effects markedly contribute to the narrowing of the structural ensembles: thermal contraction, reduced thermal motion within local potential wells, and the equilibration into lower free-energy conformations by overcoming separating free-energy barriers. During cooling, barrier heights below 10 kJ/mol were found to be overcome, which is expected to reduce B-factors in ensembles imaged by cryo-EM. Our approach now enables the quantification of the heterogeneity of room-temperature ensembles from cryo-EM structures. The rapid temperature drop during plunge-freezing affects the structural ensembles obtained by cryo-EM. To quantify the extent of perturbation, Bock and Grubmuller combined continuum calculations, MD simulations, and kinetic models.

Automated summary

Structure determination by cryo-EM provides atomic-resolution information on structural heterogeneity and ensembles. This study focuses on quantifying the effects of cooling during cryo-EM imaging by combining continuum model calculations, molecular dynamics simulations, and kinetic models. The findings suggest that thermal contraction, reduced thermal motion, and equilibration into lower free-energy conformations contribute to the narrowing of structural ensembles. This research provides a method to quantify the heterogeneity of room-temperature ensembles from cryo-EM structures.