Localized Approach to Cryo-EM CTF Estimation

The CTF analytically describes the convolution applied to the images by the electron-optical system. Estimating its properties with high precision is essential for reversing the effects and obtaining high-resolution reconstructions^{43 (link)}. Whereas the methodology for measuring defocus and astigmatism from a micrograph’s power spectrum (PS) has been well-established^{12 (link), 44 (link)}, the recent increase in EM map resolution calls for a more localized approach. Local defocus variation of a seemingly flat sample can exceed 60 nm within a single micrograph, resulting in an out-of-phase CTF for some particles at resolutions beyond 3 Å. Attempts to address this issue by fitting the defocus per-particle have been made^{14 (link)}, but they require knowledge of particle positions, and lack robustness for all but the largest particle species. Even with a local smoothing approach, per-particle defocus requires high particle density to not lose accuracy compared to a global estimate. On the other hand, strong local irregularities in the specimen surface are almost never observed in tomographic volumes in vitro^{28 (link)}, suggesting per-particle precision might be unnecessary.