Abstract
Cryo-electron microscopy (cryoEM) along with image reconstruction techniques can produce vivid images of biological macromolecules in their “native” state, although objective interpretation of these images is influenced by the fact that the contribution of phase contrast greatly exceeds that of amplitude contrast in such weakly scattering objects. The microscope contrast transfer function (CTF), which is strongly dependent on the defocus level of objective lens, modulates images of the object density distribution as a function of spatial frequency. Compensation for the effects of phase contrast transfer is important because underweighting of the low spatial frequencies usually causes difficulties in evaluating absolute mass distributions in objects.Correct compensation for the CTF is difficult to achieve. This is due, in part, to ambiguities in measuring the exact defocus level in noisy micrographs, and in knowing the relative contributions of amplitude and phase contrast, beam coherence, and inelastic scattering. The availability of atomic resolution determinations for a few viruses allows one to determine empirically how to correct the cryoEM images to best fit the x-ray data.
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