Atomic structure determination by X-ray diffraction calls for large, well-formed protein crystals. Many proteins—especially membrane proteins—cannot be coaxed into forming large crystals but will often form nano- or micrometer-sized crystals. Such tiny crystals would have been considered useless just a few years ago, as they are extensively damaged before diffraction data can be recorded, but new technologies are enabling high-quality data to be collected from them.

New technologies allow protein structures to be solved from tiny crystals. Credit: panic_attack/iStock/Thinkstock

One approach is femtosecond X-ray crystallography, in which tiny crystals are streamed into the path of femtosecond pulses from an extremely bright X-ray free-electron laser (XFEL). Because the X-ray pulses are so fast, diffraction snapshots can be collected from individual microcrystals before they are annihilated. Rapid developments since the introduction of the technique in 2011 have been expanding the range of applications. The year 2014 brought new structures and biological insights, as well as improved methods for introducing tiny crystals into the XFEL beam path and for data analysis. Last year also saw some of the first biological results from the XFEL facility in Japan. New facilities in Germany and Switzerland and upgrades to the first XFEL facility, in California, are expected in the next few years.

A newer technique may yet give these (extremely expensive) XFELs a run for their money. The MicroED method, introduced in late 2013 (eLife 2, e01345, 2013), uses electron diffraction for atomic protein structure determination from three-dimensional microcrystals. Though electron crystallography has been around for decades, technical challenges have prevented the approach from becoming widespread. A recent generalized version of MicroED (Nat. Methods 11, 927–930, 2014) exposes a microcrystal to a very low electron dose while it is continuously rotated about its axis, thereby allowing the data to be processed with software tools developed for X-ray crystallography.

It will be interesting to see whether some of the advantages attributed to XFELs can be ported to electron diffraction, which uses widely available electron microscopy instrumentation. Further developments of both X-ray and electron diffraction using tiny protein crystals are likely to have a lasting impact in structural biology.