Accessible atomic structures from sub-micron protein crystals

New instruments and techniques expand the reach of crystallography, making it more efficient and/or accessible to the greater scientific community. However, despite technological improvements, crystallography still faces outstanding challenges. As an example, structure determination from sub-micron crystals remains largely out of reach for conventional crystallography. In this issue of Acta Crystallographica Section A, the lead article by Hattne et al. (2015) outlines the steps involved in the collection and processing of data from thin (i.e. sub-micron thick) crystals by the three-dimensional electron crystallography method known as MicroED A crystallographer can use table-top or synchrotron X-ray sources to collect diffraction data from protein crystals that are microns in size or larger. These sources are constantly improving, but are not yet advanced enough for use with sub-micron protein crystals (Holton & Frankel, 2010). Smaller crystals require the use of brighter X-ray sources (Gruner & Lattman, 2015). X-ray free-electron lasers (XFELs) have helped to produce structures from nanocrystals, including an experimentally phased structure of lysozyme (Barends et al., 2014). In principle, using a bright, coherent X-ray beam and new approaches to phasing, the tiniest crystals could be used (Miao & Rodriguez, 2014) and even single protein molecules could be resolved (Miao et al., 2015). In practice, however, XFEL crystallography faces obstacles. XFELs are expensive to build, can require large quantities of sample (Boutet et al., 2012; Cohen et al., 2014), and with only two sources currently available worldwide (Emma et al., 2010; Ishikawa et al., 2012), beam time is scarce. Two years ago, a group led by Tamir Gonen determined the structure of lysozyme at 2.9 A resolution from thin, sub-micron-sized crystals at cryogenic temperatures using an electron microscope (Shi et al., 2013). A low-dose tilt series allowed them to extend electron crystallography to three dimensions. They coined the term ‘MicroED’ for their method. Since that initial demonstration, several other structures have been determined by MicroED. All have relied on molecular replacement for structure determination. Shortly after the first demonstration of MicroED, the Gonen group developed a method of continuous rotation to improve the quality of the measured MicroED intensities, once again on lysozyme crystals (Nannenga, Shi, Leslie & Gonen, 2014). Two more structures were then determined; catalase (Nannenga, Shi, Hattne et al., 2014; Yonekura et al., 2015) and calcium ATPase (Yonekura et al., 2015). Over the course of two years, use of the method has spread to multiple laboratories around the world that are equipped with different electron microscopes and detectors. The use of an electron microscope for this purpose is not entirely surprising. Electrons are the quanta of choice for structure determination of proteins using single-particle methods: they offer a larger scattering cross section than X-rays and can be precisely manipulated by electromagnetic lenses. Modern electron microscopes are engineered to visualize even single protein molecules with high spatial resolution (Cheng et al., 2015). Since the seminal studies on bacteriorhodopsin by Henderson and colleagues (Henderson et al., 1990), electrons have also been used to solve structures of proteins from two-dimensional crystals, recently at atomic resolution (Gonen et al., 2005). The method of MicroED is at heart a marriage of electron cryo-microscopy (cryo-EM) and crystallography. Crystal growth and screening for MicroED is adapted from standard crystallographic approaches (vapor diffusion and batch), using similar screens and equipment. EM grids are prepared by treating crystals in solution like a single-particle suspension, a technique borrowed from protocols well known to cryo-EM specialists. ISSN 2053-2733