Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold

Cryoelectron microscopy (cryo-EM) is emerging as a major method for elucidating the structures of proteins in atomic detail. A key limitation, however, is that cryo-EM is applicable only to sufficiently large macromolecular complexes. This places a great many important proteins of smaller size, especially those of interest for therapeutic drug development, outside the reach of cryo-EM. We describe a protein engineering effort that overcomes the lower mass limit through the development of a modular imaging scaffold able to rigidly bind and display practically any small protein of interest, greatly increasing its effective mass. We show this technology can be used to visualize molecules, such as a key cancer protein, with important implications for drug design and biomedical research.