Mechanistic Insights into Cryo-cooling Induced Conformational Biases in Protein Crystal and Solution Environments

Cryo-cooling is an indispensable procedure in X-ray diffraction (XRD) and cryo-electron microscopy (cryo-EM), yet how it modulates the protein conformational ensemble remains incompletely understood. In this study, we performed large-scale molecular dynamics (MD) simulations to systematically model the rapid cooling of two representative proteins, TRP-cage and ubiquitin, across crystal and solution environments. By analyzing hundreds of molecular copies at varying cooling rates (1, 10, and 200 K/ns), we demonstrate that cooling progressively suppresses spatial dynamics, driving the ensemble to converge toward low-energy states.
Our results reveal a differential suppression effect: structurally rigid regions undergo pronounced and systematic motional restriction, while flexible regions preserve considerable disorder due to kinetic trapping within a rugged, multi-well energy basin. We propose that the final captured ensemble after cryo-cooling is governed by the competition between cooling rates and conformational exchange kinetics. Slower cooling promotes conformational reequilibration toward global minima. Conversely, faster cooling favors kinetic trapping, effectively preserving the initial flexibility patterns within the cooled ensemble.
Furthermore, we show that conformational biases in the crystalline state are notably less cooling-rate-sensitive than those in solution, presumably because crystal packing restricts the accessible phase space and elevates energy barriers. These findings provide a mechanistic framework for understanding how vitrification reshapes the protein conformational ensemble, offering essential theoretical support for bridging the gap between static cryogenic snapshots and physiological dynamics.