Sub-3 Å apoferritin structure determined with full range of phase shifts using a single position of volta phase plate

Abstract

Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87 Å resolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5 Å resolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM.

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

“…2) is negative, both halves of the split data are affected, and so the correlation coefficient between halves is positive even if the resulting reconstruction has a negative correlation with the truth. If the coma causes very strong modulation in the FSC (11), then it is easy to recognize the correct resolution limit corresponding to the first zero of the Bessel function J 0 (Eq. 1, 2).…”

“…The problem generated by the presence of large coma was analyzed in a recent publication (11) which described the refinement of EMPIAR 10263, dataset III, with coma refinement performed by Relion 3.0 (13) and JSPR (11, 22), with results presented in Fig. 6B in (11). We reprocessed this dataset and obtained a much tighter clustering of coma values, similar to the clustering observed for our datasets.…”

“…Relion 3.0 (13) seriously underestimated coma by a factor of ∼4 compared to our estimate and the average of the JSPR individual refinements per micrograph had a value that was underestimated by twenty-five percent compared to our values. However, we used a similar target function in our refinement procedure to JSPR (11, 22) and Relion 3.0 (13), and so the different outcomes are most probably due to differing refinement schemes (11). The results presented in Fig.…”

“…The acceleration in cryo-EM data acquisition enabled by the beam-image shift method (6-9) has resulted in discussions of the best experimental strategies for data collection, approaches to compensate for or to correct for optical aberrations, and methods to assess modulation and the loss of signal due to the presence of uncorrected optical aberrations (4, 6, 10, 11). Axial coma can be corrected by applying compensating beam tilt during data collection; however, beam tilting does not correct other aberrations (4), and thus the extent to which one can apply beam-image shift without compromising data quality remains open.…”

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

“…Interestingly, most of the reconstructions resolved at a resolution of 2 Å or better correspond to apoferritin from various organisms ( Fig 1D, Fig 1E, Table S1), as a result of its high symmetry and intrinsic stability. Indeed, apoferritin is now commonly used as a standard sample to assess microscope performance and implement developments in cryo-EM [5], [9], [10], [11].…”