Postdoc @rockefeller. Making cryoEM into a tool for RNA biophysics one molecule at a time.
Miro Astore
Responses to this work have been great. I want to specifically highlight the contributions of Robert Clark tinyurl.com/5dmm5yns. He started this project as an intern and made it into a rigorous study. He's starting to look for postdocs and I cant recommend him highly enough. Please reach out to him!
It's hard to express how much work went into this in a few tweets and how excited we all are by the results. Big things are still to come from CryoEM. This work has been a true team effort so thank you to all my coauthors and the scientific computing core at the Flatiron.
We just preprinted one of my favorite studies @FlatironInst
. I was lucky to be part of an amazing team studying the effects of rapid cooling to preserve samples in cryoEM. Read on to learn about the limits of cryoEM for biophysics and how to overcome them www.biorxiv.org/content/10.6...
CryoEM relies on rapid cooling to form vitreous ice. It's crucial that this happens faster than crystalline ice forms which takes 22 μs. By finding the "hot" ensemble of a molecule and then cooling it, we determined the effects of this cooling and found ways to reverse them.
We cooled Trpcage at 7 rates, up to the slowest experimental rate where ice crystals form. We found that if a given state cannot exchange more than 5% of the total population to other states, its population is preserved after cooling. Further, no states were lost due to cooling.
Our model system was the Trpcage protein which cold-unfolds. Meaning we know that the temperature we cooled toward has a different ensemble compared to the "hot" equilibrium we started from. By constructing an MSM with MD simulations, we could study the kinetic limits of cryoEM.
To our surprise, the rate at which we cooled the system had little influence on the solvent. By nearly every measure, the surrounding water acts like bulk no matter how quickly we cool it. This means that the glassy water in cryoEM samples acts like low density amorphous water.
Everybody gave this their all so thank you for all your dedication robbieclark.bsky.social
, Louis Smith, Ryan Szukalo
, @mleighton.bsky.social
, @sykhalid.bsky.social
, Pablo Debenedetti, @pilarcossio.bsky.social
and @sonyahanson.bsky.social. This wouldn't have happened without any of you!
We also developed a reweighting approach to reverse nearly all the effects of cooling. From the second law of thermodynamics, we found narrow constraints which allowed us to nearly completely recover the equilibrium ensemble from our cooled samples.
Something interesting happened in the protein structure though. We found that after cooling, solvent exposed residues tended to exhibit greater changes than buried ones, and the whole protein compacted slightly. Whether this effects larger molecular systems remains to be seen.
