Abstract
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 Å. 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.
Highlights
Cryo-electron microscopy of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging
Some choices may be limited by the hardware configuration of the microscope, such as the accelerating voltage, the type of detector and the presence of an energy filter, while other parameters, such as support grid type, defocus range, total exposure and objective lens aperture (OLA), are decided by the researcher or the operator
Halfway through the data acquisition session, the phase plate was retracted and the acquisition continued with the conventional defocus method (Fig. 1c)
Summary
Cryo-electron microscopy (cryo-EM) of small membrane proteins, such as G protein-coupled receptors (GPCRs), remains challenging. 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 possible to routinely determine GPCR structures at resolutions better than 2.5 Å. Some choices may be limited by the hardware configuration of the microscope, such as the accelerating voltage, the type of detector and the presence of an energy filter, while other parameters, such as support grid type, defocus range, total exposure and objective lens aperture (OLA), are decided by the researcher or the operator Alone, each of these settings may have a small and seemingly insignificant contribution, but the combination of several optimal values has a cumulative effect and could lead to a noticeable improvement in map resolution and quality. We quantified the effect of defocus amount and total electron exposure by splitting one of the datasets into corresponding subsets
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