"Vitrification" is a way to freeze water so that it forms a glassy solid instead of ice crystals, locking the proteins in place and so that the "ice" reflects electrons randomly like water does, and so the only large structure redirecting electrons is the protein particles.
Protein "structure" is a slippery concept. In situ measurements, molecular dynamics simulations and basic common sense pretty conclusively show that most proteins actually have a number of conformations they access during normal biological processes. In many cases, these conformations are required for the proteins normal functions. I used to work in the de novo protein field and it was a constant source of irritation to me that what we mainly designed were crystal structures, not dynamic functional proteins.
There's lots of structural information such as conserved folds that can be gleaned from Cryo and X-ray structures. Protein dynamics is an important part but structures published with these mechanisms have been proven biologically relevant by site directed mutagenesis an almost infinite number of times now.
Thank you. Would it be fair to say that crystallized protein is always in one specific confirmation? Compared to that, would vitrification have the chance (however random) to capture protein molecules in different conformations?
Yes, a crystallized protein will always be in a single conformation, or you won't be able to see it because the electron density map will be an average of all possible conformations, and therefore meaningless.
Single particle gives you the opportunity to see different conformations, but only if the data is discrete. If there's a continuous amount of conformations (think a molecular motor that's rotating) you would need nearly infinite data to resolve a nearly infinite number of conformations. If the data is less than continuous, you can image enough particles to see all the different conformations by constructing multiple models in parallel and using 3D angular searching to bin them by what conformation they are in. This is a computationally exhausting process, however.
I hate to be that guy, but your first statement is technically incorrect: you can have discrete alternate conformations superimposed in the electron density (usually 2-3 is the most that can be resolved), and you can have different conformations of multiple copies of the molecule. (I've personally worked with both, although the differences in the second case were small.) That's not even counting ensemble-based approaches to modeling the crystal structure, although that's arguably just a different way of representing the uncertainty.
All that aside, crystallization certainly biases it towards specific conformations, which single-particle EM does not.
Didn't realize that. My experience with xray crystallography is limited. Obviously there's some variance, but I always assumed it would simply be unresolvable/disordered in that case.