The (relatively) ancient art of crystallography has had a huge impact on modern science by showing us the location of atoms within a molecule. The structure of DNA (and how it acts as an information carrier) was worked out in part by using crystallography. The first step to understanding how a protein functions is often to figure out its structure using—you guessed it—crystallography. But despite its impact, crystallography feels a bit like an old-fashioned dark art.
To get a meaningful X-ray scattering pattern, the sample has to be crystallized in significant amounts. Without this, no structure can be determined (and crystallography really would be a dark art, in the sense of not producing any data). Crystallizing materials can be very difficult; obtaining crystals of rare, naturally occurring compounds is even more difficult. Much of X-ray crystallography is therefore devoted to coaxing recalcitrant molecules to align in regular patterns.
When a Japanese group of researchers reported X-ray crystallography without crystals, I couldn't resist delving in, even if we are a couple months late to the party.
Making X-rays work for you
X-ray diffraction patterns are formed by interference. The incoming X-rays penetrate deeply into a crystal, but a tiny fraction is scattered by each atom. Since a crystal has a regular array of atoms, the scattered X-rays add up in phase in some directions, resulting in a bright spot. By analyzing the bright spots, you can figure out the repeating pattern that scattered the X-rays.
This analysis is a bit more involved for complicated molecules, but the procedure is basically the same. Imagine that we replace each molecule with a single ball. These balls are repeated at regular intervals, which scatters the X-rays to create our pattern of bright spots. If we look a bit closer and we replace the balls with the original molecule, we see that every molecule has the same orientation, and each atom has a fixed spatial relationship to its corresponding atom in the next molecule over. This internal molecular structure creates its own diffraction pattern that overlays the basic pattern. Crystallographers spend a lot of time examining the details of which spots are present, how bright they are, and how a single spot might be broken up into multiple closely spaced spots, among other details.
The essential step is obtaining a sample of the material as a single crystal with sufficient size so that even the small spots are bright enough to see. This is often the limiting factor in X-ray diffraction analysis—getting around it is what makes this new technique so interesting.
Artificial crystals to the rescue
The ingenious step here was the recognition that it's the ordered structure that's important, not the crystal part. It just so happens that crystals are the most natural way to obtain order at the atomic and molecular scale.
But in recent years, scientists have figured out how to make crystal structures that have large, regular pores. These pores aren't just regularly arranged, they also have a regular internal structure—that is, the walls of each pore are identical to those in every other pore. This means that when a molecule diffuses into these open structures and sticks to the walls, it's going to adsorb to these surfaces in the same way and with the same orientation every time. If you have enough molecules sticking in enough pores, you create an ordered array of molecules.
The diffraction pattern is more complicated because it consists of all the contributions of the material that make up the walls of the pores and the molecule that is sitting in the pores. But because the support structure is known, it can be removed, leaving only the contribution due to the molecule of interest.
The researchers proved that this worked by performing a number of structural analyses of known molecules. They then took a molecule with a partially unknown structure and analyzed it to reveal the unknown details. In order to prove that the analysis wasn't a result of the researchers retrieving a structure they already knew should be there, they did the analysis blind. The researchers performing the structural analysis had no idea what they were analyzing.
An important aspect of this work is that the X-ray analysis requires much less raw material. The sample diffuses into the porous structure, meaning that it is very spread out compared to a normal crystal, so only a few micrograms are required (compared to milligrams for single crystal X-ray analysis). The point is that there are many purification techniques that manage to produce a few micrograms of material, but it's often very difficult and time-consuming to turn it into milligrams.
The true genius is realizing that only a single molecule per pore has to be in a fixed orientation. The rest can float around doing whatever they like. The disordered molecules simply add a smooth background to the entire pattern that is easily removed. In one case, the researchers estimated that only ten percent of the molecules that were present contributed to the X-ray pattern. This means that even when the binding between the pore surface and the sample molecule is not as specific as you would like, you're still likely to get reasonable data.
The downside is the size limitation. The molecules have to be able to diffuse into the pores, and the pores aren't that large. So at the moment, this is limited to small molecules. Unfortunately, the molecules we're most interested in learning about are proteins, which are the very opposite of small. Nevertheless, I wouldn't worry about that too much. I doubt we can use the same exact methods to make pores of a much larger diameter, but there are certainly other fabrication techniques that may provide two- and three-dimensional order arrays that allow individual protein molecules to artificially crystallize.
Overall, I think this is a major step forward.
Nature, 2013, DOI: 10.1038/nature11990