Structural determination of small proteins by cryo-EM using a coiled coil module strategy.

Electron cryo-microscopy (Cryo-EM) has traditionally been used for structural determination of proteins larger than 50 kDa. Recently, various approaches, such as fusion to a scaffold or the use of DARPins-cages, have been developed to extend its application to smaller proteins. In this study, we determined the structure of the small protein target kRasG12C by fusing it to the coiled-coil motif APH2, which is targeted by several nanobodies. This method enabled us to achieve a structure with atomic details at a resolution of 3.7 Å. The kRasG12C structure was bound to the inhibitor drug MRTX849 and GDP, both clearly visible in the density map. This method is advantageous due to its ease of setup and applicability to other targets. Additionally, we investigated several other techniques that can be applied to small proteins, regardless of the presence of a terminal helix. These advancements demonstrate the potential of cryo-EM for detailed structural analysis of a wide range of protein targets, extending cryo-EM application for drug discovery.