Technical Highlight - October 2014
Short description: Continuous-rotation MicroED solves protein structures by electron diffraction of microcrystals.
A large, well-ordered crystal is a prerequisite for three-dimensional (3D) protein structure determination by traditional X-ray diffraction. Producing good crystals can be more of an art than a science, however, and many proteins have stumped crystallographers despite careful optimizations. The relatively new X-FEL technology enables crystallographers to solve a protein's structure by taking individual diffraction snapshots of a stream of thousands of microcrystals, but only two such facilities exist in the world at this time.
Electron diffraction can also be used to solve high-resolution crystal structures. Though electron microscopy instrumentation is widely available, technical challenges with the electron crystallography technique have limited the approach to a small group of specialists. This technique requires a large number of thin two-dimensional crystals that are very susceptible to radiation damage by the electron beam.
In 2013, Gonen and colleagues reported a new method called micro-electron diffraction, or MicroED. In this approach, small 3D microcrystals are used to collect electron diffraction data taken in a tilt series of still exposures using a very low electron dose. Using specialized algorithms, the researchers solved the structure of lysozyme at 2.9-Å resolution using MicroED data from just three microcrystals.
Gonen and colleagues now report an improved MicroED data collection protocol. They developed a continuous rotation method that enables the collection of the electron diffraction data as a movie, analogous to the way that data is collected using traditional X-ray crystallography. This in turn allows the MicroED data to be readily processed using MOSFLM, an established software tool developed for X-ray crystallography.
With their improved continuous-rotation MicroED approach, the authors solved the structure of lysozyme at 2.5-Å resolution (PDB 3J6K)—and with substantially better data quality than the original MicroED approach—using just two microcrystals. Given its practicality—there is no need for large crystals or large numbers of microcrystals, and the wide availability of electron microscopy instrumentation and straightforward data processing—this method is poised to be broadly applicable.