Simulations of Trp-cage miniprotein show cryo-EM vitrification shifts conformer populations only slightly
Molecular dynamics of Trp-cage cooled at seven rates, the slowest matching experiment, found small but observable population shifts and a way to correct them.
Researchers have used molecular dynamics to test how the rapid cooling in cryo-EM sample preparation changes the distribution of protein conformations. The work, published in Proceedings of the National Academy of Sciences, covers more than 50 milliseconds of simulations of the Trp-cage miniprotein, both at equilibrium and during cooling pnas.org
Proceedings of the National Academy of Sciencespnas.orgOpen source ↗. In single-particle cryo-EM the sample is plunged into a cryogen and vitrified, which takes about 22 microseconds. Until now the effect of this step on the ensemble of biomolecules was unknown.
Simulated cooling rates and water behavior
The authors simulated seven cooling rates spanning three orders of magnitude. The slowest of them match experimental rates that still result in vitrification. They examined molecular mobility and density-temperature equations of state for water with and without the protein. From these they concluded that the vitrification of water is not altered by the protein.
To track conformational changes and relate them to kinetics, the team built a Markov state model (MSM) of Trp-cage. It was based on 5.4 milliseconds of equilibrium sampling at 277 K.
Population shifts and recovery of equilibrium
MSM states whose characteristic time exceeds the duration of the nonequilibrium cooling were generally more robust to cooling artefacts pnas.org
Proceedings of the National Academy of Sciencespnas.orgOpen source ↗. In the equilibrium ensemble at 230 K some states vanish. None vanished in the ensembles produced by nonequilibrium cooling. According to the authors, the population shifts caused by cooling are small but observable.
For more labile states, which are perturbed more strongly by cooling, the authors developed a thermodynamic inference framework. It recovers equilibrium populations from the measured vitrified ensembles. On this basis they conclude that cryo-EM can serve as a reliable and accurate biophysical technique for studying biomolecular ensembles. The simulations were carried out for a single miniprotein, Trp-cage.
