Section 19.6.4.6.2. Helical particles

The basic steps involved in processing and 3D reconstruction of helical specimens include: Record a series of micrographs of vitrified particles suspended over holes in a perforated carbon support film. The micrographs are digitized and Fourier transformed to determine image quality (astigmatism, drift, defocus, presence and quality of layer lines, etc.). Individual particle images are boxed, floated, and apodized within a rectangular mask. The parameters of helical symmetry (number of subunits per turn and pitch) must be determined by indexing the computed diffraction patterns. If necessary, simple spline-fitting procedures may be employed to `straighten' images of curved particles (Egelman, 1986), and the image data may be reinterpolated (Owen et al., 1996) to provide more precise sampling of the layer-line data in the computed transform. Once a preliminary 3D structure is available, a much more sophisticated refinement of all the helical parameters can be used to unbend the helices on to a predetermined average helix so that the contributions of all parts of the image are correctly treated (Beroukhim & Unwin, 1997). The layer-line data are extracted from each particle transform and two phase origin corrections are made: one to shift the phase origin to the helix axis (at the centre of the particle image) and the other to correct for effects caused by having the helix axis tilted out of the plane normal to the electron beam in the electron microscope. The layer-line data are separated out into near- and far-side data, corresponding to contributions from the near and far sides of each particle imaged. The relative rotations and translations needed to align the different transforms are determined so the data may be merged and a 3D reconstruction computed by Fourier–Bessel inversion procedures (DeRosier & Moore, 1970).