In previous posts on this blog I’ve discussed efforts to perform NMR inside of living cells. These experiments, performed in bacteria, are primarily intended to establish whether dilute-solution experiments veridically reproduce biomolecular structures as they appear in live organisms. Now it seems that crystallography is starting to get in on the act. This week in Nature Methods, a German-American collaborative team report X-ray diffraction patterns from protein crystals grown inside cultured insect cells (1).
This is not the first time such crystals have been observed. Typically, they are associated with the expression of a protein called polyhedrin that is part of a baculovirus – the vehicle used to insert foreign DNA into these cells. Other proteins will also form crystals when fused to polyhedrin itself or parts of its DNA. However, X-ray diffraction patterns had not previously been obtained from these crystals.
The reason for this is one of scale. As you might imagine, crystals grown in living cells cannot be much larger than the dimensions of the cells themselves. Such tiny crystals will not yield usable diffraction data using ordinary techniques. Even if some diffraction data can be squeezed out of them, the crystals will be destroyed by the radiation before a full dataset can be collected.
Koopmann et al. get around this problem using serial femtosecond X-ray crystallography (SFX), a technique in which a powerful X-ray laser is focused on the tiny crystals. The super-intense beam rapidly vaporizes the protein crystal, but that intensity also produces a little diffraction data. By firing the laser at a stream of these nanocrystals, in principle a full diffraction pattern can be collected and used to determine a structure.
The authors of this study gathered the crystals by gently lysing the cells and separating the detritus with a centrifuge. Notably, this allows them to skip much of the tedious business of protein purification, which may be enough reason to use this technique even if the crystals themselves have no practical application. The authors then subjected some of the nanocrystals to SFX, gathering some diffraction data. Due to limits in dataset size, the authors were unable to solve the structure using this approach (the paper reports a structure from recrystallized protein), but the results seem to demonstrate the eventual feasibility of such a project.
Of course, if one has doubts about the biological relevance of a crystal structure, those doubts are unlikely to be assuaged just by the fact that a crystal grew inside a cell, as this is an abnormal event. There may be different benefits to this technique, however. The protein used here was a glycosylated enzyme from Trypanosoma brucei, a unicellular pathogen that causes sleeping sickness. This class of proteins can pose a special challenge for structural biology because glycosylation can interfere with crystallization, and is not readily reproduced during protein expression in bacteria. The approach of allowing crystals to form within insect cells that can accurately replicate the relevant glycosylation patterns may provide a significant advantage in attacking this type of structural problem. The approach could conceivably have similar advantages for membrane-associated proteins. This may, in turn, open up new targets for drug design.
Koopmann, R., Cupelli, K., Redecke, L., Nass, K., DePonte, D., White, T., Stellato, F., Rehders, D., Liang, M., Andreasson, J., Aquila, A., Bajt, S., Barthelmess, M., Barty, A., Bogan, M., Bostedt, C., Boutet, S., Bozek, J., Caleman, C., Coppola, N., Davidsson, J., Doak, R., Ekeberg, T., Epp, S., Erk, B., Fleckenstein, H., Foucar, L., Graafsma, H., Gumprecht, L., Hajdu, J., Hampton, C., Hartmann, A., Hartmann, R., Hauser, G., Hirsemann, H., Holl, P., Hunter, M., Kassemeyer, S., Kirian, R., Lomb, L., Maia, F., Kimmel, N., Martin, A., Messerschmidt, M., Reich, C., Rolles, D., Rudek, B., Rudenko, A., Schlichting, I., Schulz, J., Seibert, M., Shoeman, R., Sierra, R., Soltau, H., Stern, S., Strüder, L., Timneanu, N., Ullrich, J., Wang, X., Weidenspointner, G., Weierstall, U., Williams, G., Wunderer, C., Fromme, P., Spence, J., Stehle, T., Chapman, H., Betzel, C., & Duszenko, M. (2012). In vivo protein crystallization opens new routes in structural biology Nature Methods DOI: 10.1038/nmeth.1859