Though first described several decades ago, single-particle electron cryomicroscopy (cryo-EM) has been a relatively specialized technique, straddling the shadowy area of structural biology between light microscopy and X-ray crystallography. Very recent technology advances that substantially improve the resolution of cryo-EM are currently reenergizing the field.

In a single-particle cryo-EM experiment, macromolecular assemblies are frozen in a thin layer of ice and imaged with an electron microscope. Thousands to millions of images of individual assemblies must be computationally aligned and merged to arrive at a three-dimensional structure.

A clear advantage of cryo-EM over X-ray crystallography is that crystallization is not required, allowing for more native-like reconstructions. But though it has long been recognized that cryo-EM has the potential to reach atomic resolution, severe technical limitations have gotten in the way. These have included difficulties in producing sufficient amounts of sample, structural heterogeneity, radiation damage, electron beam–induced sample motion and poor camera efficiency.

New cameras for cryo-EM promise to generate atomic-resolution macromolecular structures. Credit: Marina Corral Spence

Until very recently, cryo-EM users have had two options for capturing electron microscope images: an inefficient digital charge-coupled device (CCD) camera or inconvenient photographic film. New cameras that detect electrons directly allow much faster and more efficient image collection than either previous option, addressing several of the above limitations. These direct electron-sensing cameras allow a movie of a sample to be recorded over the course of its exposure to the electron beam, permitting beam-induced motion to be computationally corrected by aligning the frames. Two papers published in 2013 using this strategy (eLife 2, e00461; 2013 and Nat. Methods 10, 584–590; 2013) powerfully demonstrated the ability to obtain near–atomic resolution structures for relatively small, asymmetrical assemblies, the ribosome and proteasome. Notably, the resolution in these studies was so good that substantially fewer images than typical were needed for structure determination.

Combined with rapidly improving sample-preparation methods, increasing automation of tedious tasks and new algorithms for data analysis, single particle cryo-EM is well-poised to enable new insights about macromolecular assemblies that are currently challenging to structurally characterize with any technique.