Post #2336606

2026-05-06 16:31 UTC

@Guillawme@fediscience.org this is a *FANTASTIC* fundamental question!! I don't know if your proposed method would work since I think fluorophore response might be temperature dependent as well. I am not a kineticist or a physical chemist, but I generally thing of proton transfers in water between things that have pKa in the 0-14 range to be borderline barrierless, so I kinda think maybe? how big are the things being vitrified? I don't know cryoEM #chemistry #pH

Replies (1)

@Guillawme@fediscience.org 2026-05-07 06:06

@GetzlerChem@mstdn.science The things being vitrified are macromolecules (proteins, DNA, RNA, by themselves or in complexes) from "small" (~30 kDa, big for a chemist I know) to very big (like whole virus particles). And they are always in an aqueous solution with at least a pH buffer system, a bit of salt, and whatever else is necessary to keep the particles "happy" (membrane proteins need detergents or lipid nanodiscs, etc.). These macromolecules have many solvent-exposed groups that can exchange protons. And in general, they denature at extreme pH values. Denaturation isn't a big problem because if proteins fully unfold, we see it during initial imaging and repeat the prep with new conditions until we see them in their native state. I am thinking more about pH-sensitive regulations: conformational changes caused by changes in pH, protonation state of small-molecule ligands causing them to bind differently, etc. Good point about fluorophores being sensitive to temperature. I know some of them work at cryogenic temps because this is how cryoCLEM works (cryo correlative light and electron microscopy: you localize targets by fluorescence microscopy first, then image at higher magnification in an electron microscope), but it could be that only a few work and the pH probes won't. I was hoping to find some literature about this.

Open ##2336607