Abstract
Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.
Highlights
In a typical cryo-EM single-particle reconstruction (SPR) experiment, some aberrations such as defocus are introduced intentionally, while others such as spherical aberration are unavoidable for a given setup (Cheng et al, 2015; Wade, 1992; Scherzer, 1936)
We provide formulae for assessing how the levels of uncorrected coma and trefoil affect the resolution of SPR, and discuss their impact on the validation statistics
CTF determination by power spectrum analysis is an inherent part of high-resolution cryo-EM SPR and provides estimates of the magnitude of symmetric aberrations (Wade, 1992)
Summary
In a typical cryo-EM single-particle reconstruction (SPR) experiment, some aberrations such as defocus are introduced intentionally, while others such as spherical aberration are unavoidable for a given setup (Cheng et al, 2015; Wade, 1992; Scherzer, 1936). The success of this approach and similar approaches requires co-alignment of the optical axes for multiple lenses, and if the alignment procedures are not properly executed, coma and trefoil may be present and affect the quality of the SPR results, and yet only manifest in specialized analyses (Uhlemann & Haider, 1998; Mastronarde, 2005; Suloway et al, 2005; Cheng et al, 2018; Zivanov et al, 2020). We restrict our discussion here to axial aberrations, but with the understanding that these aberrations will have different values in different positions of an optical system for data acquired with the beam-image shift method, except for coma, which can be compensated for by the beam tilt (Wu et al, 2019; Glaeser et al, 2011)
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