|Fig. 1: Cartoon illustration of the complex structure of the 26S proteasome. Conventional ways to study this would be to purify subunits, the many individual colors, and use NMR or X-ray crystallography to understand the structure and dynamics of each subunit. (Source: Wikimedia Commons)|
Uncovering complex 3D structures of biological macromolecules is important for understanding biological processes such as disease. Many spectroscopy methods have been developed to visualize and infer these structures, such as nuclear magnetic resonance (NMR) and X-ray diffraction; however, these methods have been limited based on the size of the complex in question.  In particular, the stringent size limits of these methods have made it so that researchers need to study smaller complexes and then infer their role in the larger complex. In the past, this has made understanding complex biomolecules, such as the proteasome (see Fig. 1), very challenging. Recent advances in electron microscopy have led to cryogenic electron microscopy (cryo-EM) elucidating large biological macromolecules at near-atomic resolution.
Electron microscopy (EM) was pioneered in the early 1930s and revolutionized how we investigate structural biology. Instead of using photons to illuminate structures like in light microscopy, EM uses an electron beam and allows for much higher resolution. However, the challenge with electron microscopy is that it disrupts the native environment of these complexes through radiation damage. In addition, to avoid electron scattering, electron microscopes must be operated in a high vacuum which made it difficult to study biological samples in their natural environment. To circumvent these challenges, researchers in the 1970s decided to first freeze these samples in a thin layer of a noncrystalline form of solid water, in a method known as cryogenic electron microscopy (cryo-EM). Cryopreserving the samples allowed for them to be stable in a high vacuum and protects against some of the effects of radiation.  This also allowed 2D imaging of many orientations, which could then be used to calculate 3D structure. The problem was that these structures were lower resolution and were limited to large complexes compared to other methods such as X-ray diffraction. 
Recent advances in technology led to digital electron detectors, which allow for the recording of movies during exposure to get even more information.  These detectors use complementary metal-oxide-semiconductor (CMOS)-based sensors which do not require electron-to-photon conversion, avoiding image degradation with previous film based methods. [2,3] This improved the signal-to-noise, or the detective quantum efficiency (DQE), of imaging and made it possible to get movies and correct for movement with image processing methods. These advancements, combined with increasing computational power, allowed cryo-EM to approach near-atomic resolution for large complexes.  This improvement has allowed for cryo-EM to visualize small-molecule compounds such as drugs for drug screening and improving drug design.  In addition, membrane embedded proteins, which are very difficult to purify for other methods, could be imaged in a lipid environment. This is important for drug studies because half of all drug small-molecules bind to membrane proteins.  Many pharmaceutical companies are heavily investing in cryo-EM for drug design and testing. 
Three scientists were awarded the 2017 Nobel prize in Chemistry for their work on cryo-EM and enhancing its crucial role for structure determination.  As of today, cryo-EM has led to over 1800 solved structures, such as the structure of the Zika virus, and is the leading method for determining structures of larger biomolecules at near-atomic resolution. 
Advancements in technology are enabling new frontiers of exploration in biology. After years of enduring the size and resolution limitations of previous technologies, cryo-EM has revolutionized our ability to explore biological macromolecules. These efforts have the potential to create more specific drug targets by understanding the binding interface of macromolecules. There are still many improvements to cryo-EM to arise and push the resolution all the way to atomic. The main challenges that cryo-EM faces are related to the time and effort sample preparation and data acquisition pose; until that challenge has been met, other methods will be used to complement cryo-EM. Nevertheless, researchers are very optimistic about the future of this technique. 
© Jeffrey Granja. The author warrants that the work is the author's own and that Stanford University provided no input other than typesetting and referencing guidelines. The author grants permission to copy, distribute and display this work in unaltered form, with attribution to the author, for noncommercial purposes only. All other rights, including commercial rights, are reserved to the author.
 X. Bai, G. McMullan and S. H. Scheres, "How cryo-EM is Revolutionizing Structural Biology," Trends Biochem. Sci. 40, 49 (2015).
 R. Fernandez-Leiro and S. H. Scheres, "Unravelling Biological Macromolecules With Cryo-Electron Microscopy," Nature 537, 339 (2016).
 Y. Cheng et al., "A Primer to Single-Particle Cryo-Electron Microscopy," Cell 161, 438 (2015).
 B. E. Bammes et al., "Direct Electron Detection Yields Cryo-EM Reconstructions at Resolutions Beyond 3/4 Nyquist Frequency," J Struct. Biol. 177, 589 (2012).
 M. Peplow, "Cryo-Electron Microscopy Makes Waves in Pharma Labs," Nat. Rev. Drug Discov. 16, 815 (2017).
 D. Cressey and E. Callaway, "Cryo-Electron Microscopy Wins Chemistry Nobel," Nature 550, 167 (2017).