Abstract
Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.
Highlights
During the course of the past 10 years, spectacular advances have been made in the ability to solve macromolecular structures using cryo-EM, culminating in the 2017 Nobel Prize in Chemistry awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for developing the technique and applying it to high-resolution structure determination of biomolecules in solution (Cheng et al, 2017)
A common problem in cryoEM protein sample preparation is incomplete wetting of the grid surface, which can be solved by adjusting the plasma and conditions to achieve a more uniform spreading of the solution on the grid (Figure 2B)
Protein sample preparation still remains a trial and error process, where different approaches have to be explored in order to maximize the chances for success
Summary
During the course of the past 10 years, spectacular advances have been made in the ability to solve macromolecular structures using cryo-EM, culminating in the 2017 Nobel Prize in Chemistry awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for developing the technique and applying it to high-resolution structure determination of biomolecules in solution (Cheng et al, 2017). In order to solve this limitation, alternative blotting-free methods that ensure a more reliable and reproducible grids preparation have recently been developed. Surfaces such as amorphous carbon, metal support structure, filter paper and air-water interface affect the way particles behave and are distributed on the grid (Figure 2B).
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