One time I shipped some protein crystals and on the shipping form it asks what’s contained in there and what the value is. I wonder what they’d say if I just put “crystals” and “priceless”…  Because they really are! X-ray crystallography can help you figure out what proteins “look like” at the near-atomic level. But you need to get the protein to crystallize….which they don’t like to do… Crystallization-wrangling can be the biggest time and resource taker, so that’s what I want to discuss today. 

I made a video about it and then just put in some text below which is adapted from a much longer post on x-ray crystallography with lots more detail. And I plan to make a more practical video on actually doing the crystallizing later. Also some figures at the end. Hope this all is useful to someone(s)!

Basically, with X-ray crystallography, you get a protein to organize into a regular repeating pattern (a lattice) which we call a crystal. In order to do that you have to get the protein really really pure and then you take that dissolved protein and (through a lot of screening for good conditions) convince the individual protein molecules to ditch their watery coats (come out of solution) in an orderly manner, swapping some of their protein-water contacts for protein-protein ones. As I’ll get into, this isn’t always easy, but once you get a crystal, you can shoot X-ray beams at it.

X-rays are “just” really energetic light waves and when they run into the electrons of the atoms making up the proteins inside the crystal, they get scattered. Kinda like dropping a ball in a pool, the electric field of X-rays perturbs the electron clouds surrounding the nuclei of atoms, causing them to deflect the X-rays (which came in as a strong “united” wave) into little “mini waves” going in all directions. And this happens in each of the lots and lots of individual protein molecules inside the crystal. Since waves have consistent properties and a crystal has a defined spacing, for each wave scattered, there’s almost always another wave exactly out of phase (e.g. one’s at a high point (peak) when another’s at a low point (trough)). As a result, most of the scattered rays will cancel each other out (destructive interference), but some will add together to give you a stronger wave (constructive interference). ⠀

A “diffraction pattern” consists of a series of spots showing us where those strong “diffracted” waves hit a detector – and then we (our computers) work backwards mathematically from those spots to figure out where the electrons are that scattered them. It’s not quite that easy because we’re still missing something called “phase” information – we can tell how strong a signal is when it hit the detector (from the intensity of the spot) but not where in the wave cycle a wave was when it hit to give you that spot (was it at a peak? A trough? Somewhere in between). We can infer phase information by using additional evidence from similar known structures (a method called molecular replacement), or by adding heavy atoms (such as mercury) and comparing the patterns you get with and without them, Heavy atoms give stronger signals and thus put “place markers” in the data to help guide us.⠀

Once we have the intensities (from the original diffraction pattern) and the phase information, we use that combined data to generate an “electron density map.” These are typically represented as a meshy-looking things. Then – since we know that electrons orbit around the dense central part of atoms (the atomic nuclei) – we can build an atomic model (those sticky or ribbony things) into that mesh indicating the location of the center of each of the atoms that make it up. ⠀

It’s usually just that final model that the science consumer sees, but there’s so much more cool science (and hard work) behind getting it. So let’s get into some more detail, starting with the crystal-making itself, as the quality of the crystal is of upmost importance. So how do we get good crystals? Well, first off, what exactly is a crystal?⠀

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 “identical tiles” in a protein crystal are called “unit cells” and they are made up of 1 or more copies of some unique part called the “asymmetric unit.” That asymmetric unit may itself contain one or more individual copies of the protein. A space group is the recipe for making one unit cell from an asymmetric unit and lattice translations are the recipes for making a whole crystal from the unit cells. Don’t worry too much about these terms (and they probably make more sense if you look at the figure). The bottom line I want you to see is that if you know what’s in the asymmetric unit and you know the layout of the crystal, you can figure out the structure of the entire crystal. And you can use the symmetrical layout of the crystal to help you figure out the structure of the asymmetric unit.⠀

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).⠀

Unfortunately, getting these crystals to form from dissolved proteins is not always easy… ⠀

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”⠀

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.  ⠀

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. ⠀

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. ⠀

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).⠀

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 from them. ⠀

We put all that effort into getting great crystals because the quality of the crystal will influence the “resolution” of the data you collect.⠀

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