The State of Biophysics - Biophysical Journal

The Current Revolution in Cryo-EM

1011

FIGURE 4 A small region from the 2.2 A˚ resolu- tion cryo-EM reconstruction of b -galactosidase ( 20 ). The resolution is high enough to see an ordered and bound water molecule in the center ( yellow ). To see this figure in color, go online.

What biological insight does one gain from higher resolu- tion? Consider Fig. 5 , which shows a b -sheet from the sheath of a type VI secretion system in Vibrio cholerae . The resolution of this cryo-EM reconstruction ( 21 ) was ~3.2 A˚ , high enough to allow a complete chain trace of ~600 amino acids in the asymmetric unit of the sheath. Because the sequences of the two proteins in this asym- metric unit were known, it was possible to thread this sequence through the density placing the large and bulky side chains (as in Fig. 3 ) into their corresponding density. If one had only 5 A˚ resolution, portions of this structure might have been built correctly but ambiguities would have existed, such as in the b -sheet shown, and it would be unlikely that the correct connectivity could be estab- lished. Or consider Fig. 4 , where ordered water molecules are visualized by cryo-EM reconstruction, and suggest that the resolution is high enough to understand enzymatic reac- tions or design drugs. The rapid advances in cryo-EM over the past several years make it nearly impossible to predict where the field will be in several years. It is reasonable to expect that the expo- nential growth of near-atomic resolution structures deter- mined by cryo-EM ( Fig. 1 ) will continue, but we still do not know possible limits in resolution or how large a com- plex must be to reach near-atomic resolution. The future of cryo-EM is certain to be exciting, with new biological in- sights gained from this powerful technique. CONCLUSIONS

and the authors suggested that the main limitation on resolu- tion that we now face may be simply the intrinsic flexibility of proteins and not the hardware or software that we use. Never- theless, it is clear that further improvements in microscopes, detectors, and image processing software will lead to many more macromolecular complexes that can be solved at resolu- tions approaching 2.0 A˚ . The growing field of super-resolu- tion light microscopy has been aimed at surmounting the fundamental limitation on resolution in light microscopy, the wavelength of light (~0.5 m m or 5000 A˚ ). In cryo-EM, the wavelength of the electrons is typically < 0.02 A˚ , there- fore the wavelength of the illumination does not set the phys- ical limit on resolution.

FIGURE 5 A region from the type VI secretion system sheath of Vibrio cholerae ( 21 ), reconstructed by cryo-EM at ~3.2 A˚ resolution. The atomic model that was built into the reconstruction ( transparent gray surface ) has three different molecules shown in cyan, red, and blue. At a resolution worse than ~4 A˚ , one might have ambiguities in tracing the individual polypeptide chains present in each molecule. The resolution that was achieved prevented any such ambiguities, and it was clear that the b -sheet shown involved two strands from one molecule ( cyan ), and one strand from each of two different molecules ( red and blue ). To see this figure in color, go online.

REFERENCES

1. Watson, J. D., and F. H. Crick. 1953. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 171:737–738 .

Biophysical Journal 110(5) 1008–1012