bioRxiv preprint – Routine Single Particle Tomography

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Alex J Noble, Venkata P Dandey, Hui Wei, Julia Brasch, Jillian Chase, Priyamvada Acharya, Yong Zi Tan, Zhening Zhang, Laura Y Kim, Giovanna Scapin, Micah Rapp, Edward T Eng, William J Rice, Anchi Cheng, Carl J Negro, Lawrence Shapiro, Peter D Kwong, David Jeruzalmi, Amedee des Georges, Clinton S Potter, Bridget Carragher
doi: https://doi.org/10.1101/230276

 

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.