Effect of fringe-artifact correction on sub-tomogram averaging from Zernike phase-plate cryo-TEM

Abstract

Zernike phase-plate (ZPP) imaging greatly increases contrast in cryo-electron microscopy, however fringe artifacts appear in the images. A computational de-fringing method has been proposed, but it has not been widely employed, perhaps because the importance of de-fringing has not been clearly demonstrated. For testing purposes, we employed Zernike phase-plate imaging in a cryo-electron tomographic study of radial-spoke complexes attached to microtubule doublets. We found that the contrast enhancement by ZPP imaging made nonlinear denoising insensitive to the filtering parameters, such that simple low-frequency band-pass filtering made the same improvement in map quality. We employed sub-tomogram averaging, which compensates for the effect of the “missing wedge” and considerably improves map quality. We found that fringes (caused by the abrupt cut-on of the central hole in the phase plate) can lead to incorrect representation of a structure that is well-known from the literature. The expected structure was restored by amplitude scaling, as proposed in the literature. Our results show that de-fringing is an important part of image-processing for cryo-electron tomography of macromolecular complexes with ZPP imaging.

The Developmental Process of the Growing Motile Ciliary Tip Region

“…Figure 4a shows that the zone approximately 50 to 400 nm proximal to the FTC is occupied by the central pair microtubules and extended singlet A-tubules. Notably, the A-tubules extended into the ciliary tip region appear undecorated, whereas axonemal spacer proteins (radial spokes and outer dynein arms) are usually visible under cryo-EM 30 – 33 . This observation, along with the absence of B-tubules suggests that the growing ciliary tip region lacks a full complement of axonemal proteins.…”

Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy

“…Currently, two types of phase plate, Zernike and Volta, are adopted for cryo-EM 26 . ZPP physically exhibits relatively better contrast transfer at low spatial frequency than the Volta phase plate (VPP) 27 , although it shows strong fringe artifacts around objects come from the center hole. ZPP cryo-EM successfully enhanced the image contrast of DDM-solubilized and ice-embedded Eh V-ATPase particles, making it possible to process the particle images.…”

The advent of structural biology in situ by single particle cryo-electron tomography

“…Most significantly, the Volta phase plate (Danev et al 2014 ) was recently used to demonstrate cryoSPT at subnanometer resolution (Khoshouei et al 2016 ), averaging merely ~1400 ribosome subtomograms. Earlier phase plates caused strong ringing or “fringe” artifacts due to the cut-on frequency determined by the size of the central hole responsible for the phase shift, necessitating cumbersome computational correction (Kishchenko et al 2015 ). Hole-less phase plates, such as the Volta phase plate, reduce such artifacts and are thus poised to revolutionize cryoET and cryoSPT.…”