Energy filtering and correction for the contrast transfer function (CTF) of frozen hydrated objects as illustrated by a reconstruction of Dictyostelium S1 decorated actin

Abstract

Ice embedded samples can not be simply described as weak phase objects, but as strong interacting specimens with inelastically dominated multiple scattering. Removal of the inelastically scattered electrons allows closer to focus imaging and improves the structural signal to noise ratio, both leading to a higher structural information content. Recent work demonstrated, that for radial density distributions the CTF correction of energy filtered images was necessary and sufficient to obtain good agreement with X-ray data. Here we present the fully CTF-corrected three dimensional structure of F-actin decorated with Dictyostelium myosin head fragment (MHF). The results show aggreement to the known protein structure after CTF correction. In addition to the radial density profile also amplitudes of Bessel terms in the Fourier Transform are changing.Actin from rabbit muscle was decorated with Dictyostelium myosin head fragment under the usual buffer conditions. Images were taken with a Zeiss EFTEM EM902. Magnification was 50000x, electron energy 80kV, electron energy loss δE=0eV (zero-loss mode), energy width ΔE=20eV, defocus values were in the range of 500-900nm. Vitrified samples were observed at a temperature of 90K. All images were taken at an specimen electron dose of 500 e−/nm2, negative material was Kodak SO163 developed for 12 min in undiluted Kodak D19. For image processing and the helical reconstruction a new set of computer programs was written.