posted on 2020-05-11, 13:34authored byY. Zhang, R. Tammaro, P.J. Peters, R.B.G. Ravelli
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.