High-resolution cryo-EM reconstructions in the presence of substantial aberrations

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.

Keywords: 3D reconstruction and image processing; automation; axial aberrations; coma; cryo-EM; imaging; resolution; single-particle cryo-EM; structure determination; trefoil; validation.

Figure 1

Resolution dependence of third-order aberrations on single-particle reconstruction. The x axis represents reciprocal-space resolution, scaled to a = 1 for x = 1 (equation 1). The y axis represents reciprocal-space signal modulation of the reconstruction resulting from averaging the aberration. Three values of aberration coefficients are shown here: black is 1, blue is 0.5 and orange is 0.25.

Figure 2

Each row corresponds to one of our three experiments performed with high coma (beam-tilt) values. The left panel shows oscillations of amplitudes caused by uncorrected aberrations calculated with (1) and (2) for each experiment, the middle panel shows FSC plots before (black) and after (red) coma correction and the corresponding final map fragment for three experiments with high coma (beam-tilt) values. The statistics from each experiment are presented in Table 1 ▸. The vertical dotted gray line represents the first zero of the modulation function (2) and the solid line represents the first zero of the modulation function with the assumption that the coma impact was compensated by image translation (5). The resolutions corresponding to the first zero values are listed in Table 2 ▸. The right panel shows fragments of unsharpened maps corresponding to the reconstructed maps.

Figure 3

Heat maps for coma and trefoil values refined separately per image from the HemQ-57K data set. The leftmost panel shows tight clustering of the coma values, with the center panel magnifying the region of clustering. The right panel shows the values of trefoil, which for this data set were very low and very consistent across the entire data collection. Dividing coma expressed in micrometres by a factor of 8.1 converts it to the equivalent mrad beam-tilt units assuming C _s = 2.7 mm.