Intro to protein crystallization (the actually getting crystals stuff and why it's so hard)

A crystal is “just” an orderly 3D lattice of repeating units - kinda like floor tiles but in 3 dimensions - if you know where one spot is on one thing you know exactly where in space the corresponding spot is in every other copy of the thing because there’s a “recipe” to follow - like stick one copy down, take 2 steps left and 3 up, stick another copy down, etc. ⠀
The reason why diffraction patterns from crystals are a series of distinct spots is because of a crystal’s symmetry. This symmetry is discussed in terms of comparing the asymmetric units - so for example an atom in one protein molecule is in the same place in its “asymmetric unit” as the corresponding atom in the protein molecule copies in each asymmetric unit. So even if the protein itself is wildly unsymmetrical (often the coolest ones are), you still have symmetry. So you’ll still get evenly-spaced wave scatterers leading to diffraction (the situation when waves constructively interfere to give a stronger signal).⠀
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Unfortunately, getting these crystals to form from dissolved proteins is not always easy…
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When something is dissolved, each molecule has a full coat of water, but to crystallize, something (like our protein) needs to come out of solution. This means it needs to “prioritize” contacts to things other than water - like other protein molecules. So, for instance, it swaps some of the water molecules it was coated in, for interactions with other protein molecules. But the tricky part about crystals is that, while they represent optimal packing layouts, they require coordination because all the molecules have to arrange themselves the same way. And if they do it in different ways you just get clumpy protein “aggregate”⠀
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Coordination takes time (think putting together a jigsaw puzzle “properly” vs just tossing all the pieces into a box). So if you don’t give molecules time to coordinate, they can’t crystallize. We can therefore use speed-up tactics to prevent crystallization when we don’t want it to occur, such as when we’re storing protein after purifying it and don’t want the water in and around the protein to crystallize and damage our protein. To prevent that unwanted water crystallization we can “flash freeze” our protein by dunking tiny tubes of it into liquid nitrogen to rapidly cool them, preventing the formation of ice crystals. ⠀
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But with X-ray crystallography, the situation’s different - we want crystallization (but of our protein and not of water!) so we want to slowly promote grouping together. ⠀
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How do we slow things down? There are a lot of different techniques for doing this. The one that I’ve used the most is “vapor diffusion,” mostly “hanging drop” crystallization. Basically you stick a drop of liquid containing your protein on a glass slide and then you flip the slide over and use it as the “roof” for a well of protein-less liquid (reservoir). Since this reservoir liquid is more concentrated than the drop liquid (because the reservoir hasn’t been diluted with your protein), water evaporates from the drop to help “dilute out” the reservoir (that’s not really its goal - it’s trying to escape the well altogether but there’s a lid, so it gets pulled in by the reservoir). This leaves less water available to surround the protein molecules. So the protein molecules start binding to each other instead - hopefully in the coordinated fashion that leads to nice crystals. ⠀
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In addition to simply removing water, we promote crystallization by optimizing the pH (acidity), salt types & concentrations, protein concentrations, etc. When you see “optimize” think “troubleshooting” and LOTS and LOTS of “trial and error” - since each protein is different and has different binding opportunities to offer up to other protein molecules and different types of interactions are favored in different conditions, the ideal “crystallization cocktail” varies from protein to protein and is normally unpredictable. Therefore, we usually carry out extensive screening - we even have liquid dispensing robots to help us do this with tiny tiny volumes so that we can test hundreds of combinations without needing a ton of protein (you still need a lot of protein though because it needs to be at a high concentration so the molecules can find one another okay - and this can be a major limitation of crystallography).⠀
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We also have a microscope robot that takes pictures of our crystal trays for us over time so that we can see if crystals are forming in any of the wells. If we get any hits, we can then optimize around those conditions in order to get even better crystals. Once our crystals have grown and stop growing (could be days to weeks to months depending on the crystal) we have to fish them out with little loops, freeze them with cryoprotectants, and store them in liquid nitrogen dewars (basically really insulated giant thermoses) to keep them super cold until we’re ready to collect diffraction data.
more: bit.ly/xraycrys...

[@wiktoriaaqaq]

Thank you!

[@hazemdarwish3659]

If my initial purified recombinant protein was saved in a concentration of 2mg/ml in size exclusion buffer with 50% Glycerol in -20°C...I have in total 5 ml therefore a total of around 10 mg of proteins.
can I dilute this and reconcentrate using the protein concentrator falcons to get rid of glycerol and then crystalize ? or should I make a new protein and make the crystallography directly without saving in glycerol stocks?
thank u

[@thebumblingbiochemist]

That completely depends on the protein. Some are okay with it, some aren't. You'll just have to try sorry!

[@terraint3697]

Has anyone ever seen atoms, proton, electrons, or neutrons other than in a cartoon?

[@thebumblingbiochemist]

Not sure what you mean by "seen" - i mean we can see evidence