Radiation Damage in Cryo-EM

Cryo-EM has revolutionized the field of structural biology, especially for large protein complexes that proved difficult for structural determination using X-ray crystallography. This is markedly true for the field of photosynthesis research, where many of the interesting targets are cofactor-rich membrane proteins around 1 MDa in molecular weight. A challenge in cryo-EM, however, is that the electron radiation delivered during the experiment damages the sample as a function of exposure time. Because cryo-EM data are collected as a series of multi-frame movies, radiation damage accumulates, which has been nicely characterized (Fig. panel A). To alleviate this, dose weighting is applied where earlier frames are weighted heavier than later frames, allowing for the preservation of high-resolution information.

Recently, the structure of the water-splitting enzyme, Photosystem II, from the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 was determined using single-particle cryo-EM to a global resolution of 1.93 Å. The extremely high resolution achieved allowed the structure to be viewed in fine detail. However, a close analysis of the metal ions in the cluster within the active site, which catalyzes the water oxidation reaction, suggested that the metals were either 2+ or 3+ rather than the expected 3+ or 4+ (Fig. panel B, see publication below). This limits the ability to perform chemical analyses of the active site based on the structure. Importantly, it also suggests that metal clusters are damaged in cryo-EM faster than amino acids. Thus, it is vital to explore how metallocofactors are damaged in cryo-EM, so that structures represent their native state. Although this has begun to be addressed (Fig. panel C), collecting cryo-EM data that can be reconstructed to high resolution using very low electron radiation dose is required to account for damage.