Better Cryo-EM Specimen Preparation: How to Deal with the Air–Water Interface?

[Display omitted] •We outline the nature and impacts of air–water interface (AWI) during cryo-EM specimen preparation.•AWI poses risks of unpredictable particle distribution, protein denaturation, complex dissociation and preferential orientation.•The review overviews various strategies to deal with the AWI, including additives in solution, fast vitrification devices and the adoption of supporting films.•Bioactively functionalized supporting films exhibit strong interaction with target macromolecules, helping to avoid the problems that the AWI causes. Cryogenic electron microscopy (cryo-EM) is now one of the most powerful and widely used methods to determine high-resolution structures of macromolecules. A major bottleneck of cryo-EM is to prepare high-quality vitrified specimen, which still faces many practical challenges. During the conventional vitrification process, macromolecules tend to adsorb at the air–water interface (AWI), which is known unfriendly to biological samples. In this review, we outline the nature of AWI and the problems caused by it, such as unpredictable or uneven particle distribution, protein denaturation, dissociation of complex and preferential orientation. We review and discuss the approaches and underlying mechanisms to deal with AWI: 1) Additives, exemplified by detergents, forming a protective layer at AWI and thus preserving the native folds of target macromolecules. 2) Fast vitrification devices based on the idea to freeze in-solution macromolecules before their touching of AWI. 3) Thin layer of continuous supporting films to adsorb macromolecules, and when functionalized with affinity ligands, to specifically anchor the target particles away from the AWI. Among these supporting films, graphene, together with its derivatives, with negligible background noise and mechanical robustness, has emerged as a new generation of support. These strategies have been proven successful in various cases and enable us a better handling of the problems caused by the AWI in cryo-EM specimen preparation.