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Free keywords:
TRANSMISSION ELECTRON-MICROSCOPY; CONTRAST CRYOELECTRON MICROSCOPY;
MOLECULAR ARCHITECTURE; RADIAL SPOKES; UCSF CHIMERA; IN-SITU; FLAGELLA;
VISUALIZATION; EM; RECONSTRUCTIONSElectron tomography; Zernike phase-plate; Sub-volume averaging; Image
processing;
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. (C) 2015 Elsevier Inc. All rights reserved.
