CTF determination and correction for low dose tomographic tilt series.
The resolution of cryo-electron tomography can be limited by the first zero of the microscope's contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction.
Xiong
Quanren
Q
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Morphew
Mary K
MK
Schwartz
Cindi L
CL
Hoenger
Andreas H
AH
Mastronarde
David N
DN
D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D012984:Software