I realize that I was just talking about crystal formation here the other day, but there’s yet more news in the area, and it comes in the fiendishly difficult area of protein crystallography. All you have to do to appreciate the horrors of this field is to step into a lab that does it for a living: stacks and stacks and stacks of plates, each with a slightly different set of conditions (buffers, concentrations, additives, protein construct, and son on) to try, once again, to get a protein crystal that diffracts well enough for structure determination. It’s especially tricky to get the ones with your small molecule sitting inside them at its binding site, but when you do, the payoffs can be huge.
Producing such things at the quality levels needed, though, is an absolute black art. There are trends (sometimes), better ideas about starting points (sometimes), rational modifications of the protein sequence to increase your chances (sometimes), but in the end, it still comes down to relentless empirical try-it-and-see. This latest paper (writeup by C&E News here) is looking at a well-known protein crystal (glucose isomerase) that is also well known to exist it two polymorphic forms (see that last post on the subject for more on polymorphs). What isn’t well known is why it forms these particular two types of crystals and how that happens. In virtually every case of polymorphism, we have little or no clue about such things, and I’m very glad to see clues starting to be unearthed.
What these authors have done is to start the crystallization process in ammonium sulfate solution and then freeze the solutions (liquid nitrogen) at various time points, imaging the crystals thus found by electron microscopy. And there are some surprises. At the twenty-second time point, there are already “nanorod” crystals forming, which are, on the average, two protein molecules wide and twelve molecules long. You are not going to get too much earlier in the crystal formation process than that. The concentration of the nanorods increases over time. However, their length does not: they seem stuck at this size. They do start to associate into fibrils as time goes on.
But there are other things happening as well. There are other species in there (trimer, tetramers, single-file chains), and after about thirty minutes you can also see faceted nanocrystals of the two known bulk polymorphs, one prismatic and rodlike and one rhombic. The smallest of each are in the 100 nM range along their shortest edges. Trying the experiment using PEG instead of ammonium sulfate (another known set of conditions for producing these protein crystals) shows that the useful concentration range for the additive is much narrower, but in both cases, at the low end of the range you get rhombic, and at the high end you get prismatic. With PEG, though, if you keep pushing it, you get no crystals at all, but rather an amorphous gel-like state. And in none of the PEG experiments did they ever observe the nanorods seen in ammonium sulfate.
That gelation pathway seemed to involve fibril formation as well, which looked in the earlier experiments like it might be a precursor to formation of the rhombic crystals. Weirdly, the authors found that you can take some of that gel and use it to nucleate another protein solution, one that would otherwise form only the rhombic crystals, and form the prismatic crystals with it instead (which is a bizarre thought to me, and probably to a lot of other people who don’t crystallize things for a living). You actually see the crystals growing out of the surface of the lump of gelatinous protein gorp, as shown at right. This is order out of chaos, for sure; I’ll bet that the temperature of the solution at that interface is changing just to make the thermodynamics of all that loss of entropy work out.
Seeing the early stages of crystal formation like this allowed the authors to come up with all the plausible models of the nanorods and compare those to experimental data. One clearly matches better than the rest of them, so now we know what the earliest crystal formation contacts are, and can start to explain why. The preferred mode of contact buries a lot more negative charges in the protein surface, and the paper goes into detail about how this can explain the effects of sulfate ions in the solution versus PEG.
Overall, it appears that the prismatic crystals (from ammonium sulfate) are made up of bunches of nanorods, rather than growing molecule-by-molecule, which is also contrary to the mental pictures that many of us have. Meanwhile, the rhombic crystals grow more like the traditional picture, monomer by monomer. The nanorod form, though, can also be trapped in a metastable fibril state, starting off as individual fibrils and going on to a gel, with too high an activation energy to dissociate and form prismatic crystals. But its surface can nucleate the formation of such crystals (as in the above photo). At really high PEG concentrations you get a different gel, which is not composed of fibrils, and seems to be a complete dead end.
What they don’t see any evidence for, though, is another mode predicted by theory (and which I mentioned in the last post on this stuff): formation of phase-separated nanodroplets that then crystallize. Those nanodroplets take us back to the hot topic of protein condensates inside cells, and perhaps its a good thing that they’re not invariably precursors to forming protein crystals (!) That is very likely the reason that such condensates seem to feature intrinsically disordered proteins as well, to avoid the crystallization problem. . .