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The combination of high-end cryogenic transmission electron microscopes (cryo-EM), direct electron detectors, and advanced image algorithms allows researchers to obtain the 3D structures of much smaller macromolecules than years ago. However, there are still major challenges for the single-particle cryo-EM method to achieve routine structure determinations for macromolecules much smaller than 100 kDa, which are the majority of all plant and animal proteins. These challenges include sample characteristics such as sample heterogeneity, beam damage, ice layer thickness, stability, and quality, as well as hardware limitations such as detector performance, beam, and phase plate quality. Here, single particle data sets were simulated for samples that were ideal in terms of homogeneity, distribution, and stability, but with realistic parameters for ice layer, dose, detector performance, and beam characteristics. Reference data were calculated for human apo-ferritin using identical parameters reported for an experimental data set downloaded from EMPIAR. Processing of the simulated data set resulted in a value of 1.86 Å from 20 214 particles, similar to a 2 Å density map obtained from 29 224 particles selected from real micrographs. Simulated data sets were then generated for a 14 kDa protein, hen egg white lysozyme (HEWL), with and without an ideal phase plate (PP). Whereas we could not obtain a high-resolution 3D reconstruction of HEWL for the data set without PP, the one with PP resulted in a 2.78 Å resolution density map from 225 751 particles. Our simulator and simulations could help in pushing the size limits of cryo-EM.

Could Egg White Lysozyme be Solved by Single Particle Cryo-EM?