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Biophys Rep. 2017;3(1):1-7. doi: 10.1007/s41048-016-0026-3. Epub 2016 Jul 22.

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

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1
Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 USA.

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.

KEYWORDS:

Air–water interface; Cryo-EM; Protein denaturation; Sample preparation

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