Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports

Abstract

Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.

Structural basis of kainate subtype glutamate receptor desensitization

“…A volume of 3 μL was added to R1.2/1.3 holey carbon grids (Quantifoil) rendered hydrophilic with self-assembled monolayer functionalization 27 , and grids were frozen with a Vitrobot Mk IV robot (FEI Company, Hillsboro, OR, USA) or a Leica EM GP (Leica Microsystems Inc., Buffalo Grove, IL, USA).…”

Overview and future of single particle electron cryomicroscopy

“…Finally, it is possible that there are other types of deleterious beam-induced specimen motion that we have not yet identified, or other technical problems that cause blurring of cryoEM images due to specimen charging. New types of specimen support will help to overcome some of these problems [29,30,26].…”

A new method for vitrifying samples for cryoEM

“…We have shown that nanowires will grow on a layer of copper of ~80nm thick and that the gold surface remains free of nanowires and ready for substrate adherence. This approach will also have the advantage that we can then provide gold substrate on a gold mesh that has been shown to be effective in reducing beam induced movement (Meyerson et al, 2014, Russo and Passmore, 2016) although it remains to be seen what effect the metal sandwich construction will have on beam induced movement.…”

“…gold, copper, molybdenum etc.) (Ermantraut et al, 1998) (Quispe et al, 2007) (Meyerson et al, 2014) (Russo and Passmore, 2016). The thin vitrified layer of sample is commonly produced by pipetting a small volume of sample (typically 2-3μl) onto the surface of a freshly plasma treated grid, and then removing most (>99.99%) of the sample using filter paper to leave a thin wet layer (typically ~100’s nm thick) across the surface.…”