Routine single particle CryoEM sample and grid characterization by tomography

TLDR

Single particle cryo‑electron microscopy is routinely performed under the assumption that particles remain free of air‑water interface adsorption and reside in thin, vitreous ice. The authors aimed to test this assumption by applying fiducial‑less tomography to more than 50 cryo‑EM grid/sample preparations to map particle distribution and ice geometry within grid holes. They used fiducial‑less cryo‑electron tomography to determine ice thickness, identify optimal single‑particle collection areas, assess particle heterogeneity, and generate de novo models for template picking and alignment. The analysis showed that roughly 90 % of particles adsorb to air‑water interfaces, a result with wide‑ranging implications for protein denaturation, conformational change, and preferred orientation, and confirmed that the tomography approach can guide ice‑thickness assessment and optimal data‑collection strategies.

Abstract

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to 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 1000 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.