Routine sub-2.5 Å cryo-EM structure determination of GPCRs

Abstract Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. Pushing the performance boundaries technique requires quantitative knowledge about contribution multiple factors. Here, we present an in-depth analysis and optimization main experimental parameters in cryo-EM. We combined actual structural studies with methods development to quantify effects Volta phase plate, zero-loss energy filtering, objective lens aperture, defocus magnitude, total exposure, grid type. By using this information carefully maximize performance, it is now possible routinely determine GPCR structures at resolutions better than 2.5 Å. The improved fidelity maps enables building atomic models will be crucial for future expansion cryo-EM into structure-based drug design domain. guidelines given here are not limited GPCRs can applied directly other proteins.