Opinion: hazards faced by macromolecules when confined to thin aqueous films

Abstract

Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air–water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air–water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions. One possible way to do so might be to preemptively form a surfactant monolayer over the air–water interfaces, to serve as a structure-friendly slide and coverslip. An alternative is to immobilize particles of interest by binding them to some type of support film, which—to continue using the analogy—thus serves as a slide. In this case, the goal is not only to prevent the particles of interest from diffusing into contact with the air–water interface but also to increase the number of particles seen in each image. In this direction, it is natural to think of developing various types of affinity grids as structure-friendly alternatives to thin carbon films. Perhaps ironically, if precautions are not taken against adsorption of particles to air–water interfaces, sacrificial monolayers of denatured protein may take the roles of slide, coverslip, or even both.

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

“…This modification was undertaken in an attempt to reduce the time the sample has to interact with the air-water interfaces in the thin liquid film prior to vitrification. These interactions are assumed to be the cause of preferred particle orientations 39 , and indeed the faster plunging speed resulted in grids that displayed markedly better particle distribution (paper in preparation), allowing for the collection of data at zero degrees tilt. Nanowire based, self-blotting grids with a lacey carbon supporting substrate were used for this purpose, following the same sample preparation methods described above.…”

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

“…The idea that particles are randomly oriented in ice after grid blotting and rapid cryo-plunging is far from the experimental reality. During the vitrification process, particles diffuse and interact with the water-air and/or water-support interfaces even 1000 times a second 15,16 . This can lead to protein denaturation but also, in almost all instances, particles are adsorbed to such interfaces and present preferential orientation due to their surface properties 17 .…”

Protein dynamics regulate distinct biochemical properties of cryptochromes in mammalian circadian rhythms

“…Some anisotropy was observed in our cryo-EM density, arising from particles adopting two preferred orientations in vitrified ice ( Figure S6), which was detrimental to the resolution of our 3D reconstruction and likely leads to an inflated value for our final map. This is likely arising from interaction with the air-water interface with our complex, as commonly seen in other protein samples analyzed by cryo-EM (Glaeser, 2016;Glaeser and Han, 2017;Noble et al, 2018). Attempts to improve the particle distribution by addition of detergents to the sample or by stage-tilting during data collection (Tan et al, 2017) were ineffective and did not result in a higher quality map.…”