Reducing the effects of radiation damage in cryo-EM using liquid helium temperatures

The physical limit in determining the atomic structure of biological molecules is radiation damage. In electron cryomicroscopy, there have been numerous attempts to reduce the effects of radiation damage by cooling the specimen beyond liquid-nitrogen temperatures, yet all failed to realize the potential improvement for single-particle structure determination. We have identified the physical causes of information loss at liquid-helium temperatures, and overcome them using a combination of nanoscale electron beam illumination and a gold specimen support with 100 nm diameter holes. This combination allowed structure determination where every frame in the exposure contained more information than was available with cryomicroscopy at liquid-nitrogen temperatures, matching expectations from crystal diffraction. Since a 100 nm hole is smaller than the field of view of a typical micrograph, the edges of the foil are directly visible in each micrograph. Protein molecules that are degraded tend to aggregate at the edges of foil holes and can constitute a significant fraction of the micrograph. This and the need for minimal water-foil irradiation will both be important to consider as new cryomicroscopes and specimen supports are developed for imaging molecules at extremely low temperatures where the effects of radiation damage are reduced.