Cooling fast and slow: Characterizing the effects of vitrification in cryo-EM and the subsequent recovery of equilibrium populations.

For the Trp-cage miniprotein, Markov State Model states with characteristic times longer than the nonequilibrium cooling duration are more robust to vitrification artifacts, and none vanish after rapid cooling.

• Why it matters: Cryo-EM requires vitrification, but how fast versus slow cooling perturbs biomolecular ensembles is unclear, limiting confidence in ensemble measurements from vitrified samples.
• What they did: Using molecular dynamics simulations over 50 ms, the authors tested seven cooling rates spanning three orders of magnitude (slowest matching experimental rates) and built an MSM from 5.4 ms of equilibrium sampling at 277 K.
• The result: They found water vitrification is unaltered by the protein, observed equilibrium states vanishing at 230 K only in equilibrium, and introduced thermodynamic inference to recover equilibrium populations from vitrified ensembles.

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