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

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

Automated summary

There is a need to optimize cryo-EM data acquisition approaches to improve the resolution of GPCR cryo-EM structures to better than 2.5 angstrom, in order to use them for structure-based drug design purposes. The authors present a systematic analysis of the main cryo-EM experimental parameters using three GPCRs as test cases, which is also of interest for the cryo-EM structure determination of other small membrane proteins.