Development of imaging scaffolds for cryo-electron microscopy

Following recent hardware and software developments, single particle cryo-electron microscopy (cryo-EM) has become one of the most popular structural biology tools. Many targets, such as viruses, large protein complexes and oligomeric membrane proteins, have been resolved to atomic resolution using single-particle cryo-EM, which relies on the accurate assignment of particle location and orientation from intrinsically noisy projection images. The same image processing procedures are more challenging for smaller proteins due to their lower signal-to-noise ratios. Consequently, though most cellular proteins are less than 50 kDa, so far it has been possible to solve cryo-EM structures near that size range for only a few favorable cases. Here we highlight some of the challenges and recent efforts to break through this lower size limit by engineering large scaffolds to rigidly display multiple small proteins for imaging. Future design efforts are noted.