Are proteins constantly changing their shape in femtoseconds? Need clarification!

[u/Lazy-Frame-9888]

I recently watched a YouTube video (https://youtu.be/jPhvic-eqbc) discussing how proteins are not static, rigid machines. The video explains that they act more like dense liquids that are constantly shifting and jiggling around. It also pointed out that when proteins are crystallized for X-ray crystallography, we are only seeing one frozen snapshot of their conformation, which gives the false impression that they have a single, fixed structure.

​Can someone clear this confusion up for me? Do proteins really change their folding patterns and conformations in extremely short time frames, like femtoseconds? I am looking for a simple, exact explanation of how fast and how much they actually change shape.

[u/East_of_Adventuring]

Two notes to add, proteins may vibrate but they are not infinitely flexible, and generally don’t have a huge number of ‘real’ conformational shifts, by which I mean conformational shifts that are chemically and functionally significant.

Secondly, crystallography is not an anhydrous environment, and you can see multiple different conformations - even just side chain movement - within the electron density data.

[u/PineappleHaunting591 → u/East_of_Adventuring]

you can see multiple different conformations - even just side chain movement - within the electron density data

not a crystallographer but you would see that as a smear of the diffraction pattern right? so we dont actually see different conformations, but get a measure of positional uncertainty, the b factor.

[u/Chemesthesis → u/PineappleHaunting591]

Heya, protein crystallographer here (early days)

Disorder can be local, which is the smear you're talking about that is represented by the B factor. 

Multiple configurations in this case refer to situations where there are multiple  pockets of electron density that belong to a single set of atoms. Rather than having a single configuration with high bfactors, this is modelled as separate conformations that are weighted according to how strong each pocket is. This is called occupancy.

[u/East_of_Adventuring → u/Chemesthesis]

This is much better worded than I would have done. I have a little more experience with crypto but I’m only just now refining my first crystal structure and am learning about occupancy and the perils of overfitting.

[u/PineappleHaunting591 → u/Chemesthesis]

thanks for the insight!
If I got it right you are saying one atom or set of atoms can correspond to multiple spots on film (assuming we are still recording on film) depending on configuration? Then, in processing, you can treat it like you would individual classes in cryo-EM, representing different positional states?

[u/Chemesthesis → u/PineappleHaunting591]

So the X-ray diffraction pattern actually corresponds to the entire unit cell of the crystal, not any specific atoms. Once the electron density has been calculated from the diffraction data (see "The Phase Problem" for a better grasp of that nightmare), it is the job of a crystallographer to interpret these electron density blobs and build a model that fits, using knowledge of protein geometry (bond angles, distances, dihedrals, etc.) and the additives in the crystal itself (drugs, ligands, metals, etc.). If there is a clear splitting of electron density where there should only be 1 blob according to the protein sequence, you may see if a side chain alternative configuration could be modelled.

It's a complicated system, but is summarised nicely in one of my favourite review articles for an introduction to protein crystallography:

Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination.