# Simulations of Trp-cage miniprotein show cryo-EM vitrification shifts conformer populations only slightly

- Published: 2026-11-05 03:31 UTC
- Section: Life & Health › Biology & biochemistry
- Tags: cryo-EM, vitrification, Trp-cage miniprotein, molecular dynamics simulation, Markov state model, cooling rate, conformational ensemble, equilibrium populations, vitreous ice
- URL: https://realtech.media/news/rt-6dcb6142c991a288ae6b7126_simulations_of_trp/

**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.**

![Simulations of Trp-cage miniprotein show cryo-EM vitrification shifts conformer populations only slightly](https://sgzwpqqelzmajkcubgkl.supabase.co/storage/v1/object/public/rt-media/2026/11/14645b4fd59dda04.jpg)
*Yitzilitt, Wikimedia Commons, CC BY-SA 4.0*

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 [1]. 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 [1].

## 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 [1]. 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 [1].

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 [1].

## 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 [1]. In the equilibrium ensemble at 230 K some states vanish. None vanished in the ensembles produced by nonequilibrium cooling [1]. According to the authors, the population shifts caused by cooling are small but observable [1].

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 [1]. On this basis they conclude that cryo-EM can serve as a reliable and accurate biophysical technique for studying biomolecular ensembles [1]. The simulations were carried out for a single miniprotein, Trp-cage.

## Sources

1. [Proceedings of the National Academy of Sciences](https://doi.org/10.1073/pnas.2619560123)

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Source: RealTech, realtech.media — https://realtech.media/news/rt-6dcb6142c991a288ae6b7126_simulations_of_trp/