Protein Engineering-Enabled Cryo-EM Investigation of Small GTPases

• Overcomes the size limitation of cryoelectron microscopy applied to small proteins. • Protein engineering to stably link two proteins via terminal helices. • Enables cryo-EM studies of RAS small GTPases and their switch regions. • Epitope mapping of a NRAS-specific monobody inhibitor. • Monobody additive minimizes preferred orientation in cryo-EM samples. Small GTPases play important roles in cellular signaling. Due to their small sizes (∼21 kDa), structural studies of small GTPases have been predominantly performed using x-ray crystallography in which crystal lattice contacts made it challenging to define unperturbed conformations of the key switch regions. Here, we develop a protein-engineering strategy that enables cryo-EM analysis of small soluble proteins and applied to RAS. We fused the C-terminal α5 helix of the RAS globular domain to a small protein BRIL by forming a continuous helix, which leaves most RAS surfaces exposed to the solvent and unperturbed, followed by the complex formation with an anti-BRIL Fab. This engineered complex with an increased molecular weight, termed “RAS-lollipop”, enabled single-particle cryo-EM of RAS. Using this approach, we determined the cryo-EM structure of NRAS, whose structural studies using crystallography have been the least successful among the RAS isoforms. We revealed the conformations of the switch region and α 5 helix that differ from those observed in published crystal structures, and also defined the binding site of an NRAS-specific monobody. We uncovered an unexpected surfactant-like property of this monobody, which reduces orientation biases of particles on cryo-EM grids. Together, this work establishes a platform for visualizing small GTPases and potentially other small proteins with minimal perturbation of their surfaces.