Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Abstract

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.Contributions:

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

“…To facilitate direct comparison, we collected tomograms from regions of matching ice thickness on grids prepared using a Vitrobot. Consistent with previous reports [36][37][38] we observed a pronounced bi-phasic distribution of sample due to clustering at the air-water interfaces when prepared on a Vitrobot (Figure 4D) among multiple areas of independently produced grids (Figure 4E). Importantly, following the same analysis and quantifications of sample grids prepared by trEM we observed a significantly more uniform distribution of protein along the z-direction (Figure 4F, G), indicating an important improvement to sample quality.…”

“…To assess the sample quality obtained by this method we first performed tomographic reconstructions to determine the protein distribution relative to the air-water interfaces [34][35][36] .…”

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

“…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 . While supplementation of buffers with surfactants such as detergents is often employed to improve particle distribution in ice 18 , for sensitive specimens including membrane proteins this approach should be avoided.…”

Automated cryo-EM sample preparation by pin-printing and jet vitrification

“…Proteins tend to absorb to the air-water interface, referred to as "the deadly touch" 12 . It seems intuitive that reducing the time the sample is exposed to such an interface would help to prevent protein denaturation 17 . However, using the Stokes Einstein equation, it has been calculated that even for a minimal residence time of ~1 ms, particle-surface interactions will occur dozens of times before the water is frozen 30,31 .…”

“…During this dynamic process, air-water interfaces will form 5,12,13 , which can be detrimental to the structure of interest as proteins tend to absorb to such interfaces and (partly) denature 12 . The resulting film of sample on a perforated carrier [14][15][16] has a generally concave shape 12,17 due to drying and draining, with the center being the thinnest. The grid is held by tweezers and plunged into a bath of cryogen to vitrify the sample, so that it can be observed in the vacuum of the microscope.…”