A strategy for determining the orientations of refractory particles for reconstruction from cryo-electron micrographs with particular reference to round, smooth-surfaced, icosahedral viruses

Cryo-electron microscopy and three-dimensional image reconstruction are pow erful tools for analyzing icosahedral virus capsids at resolutions that now extend below 1 nm, However, the validity of such density maps depends crit ically on correct identification of the viewing geometry of each particle i n the data set. In some cases-for example, round capsids with low surface r elief-it is difficult to identify orientations by conventional application of the two most widely used approaches-"common lines" and model-based itera tive refinement. We describe here a strategy for determining the orientatio ns of such refractory specimens. The key step is to determine reliable orie ntations for a base set of particles, For each particle, a list of candidat e orientations is generated by common lines: correct orientations are then identified by computing a single-particle reconstruction for each candidate and then systematically matching their reprojections with the original ima ges by visual criteria and crosscorrelation analysis. This base set yields a first-generation reconstruction that is fed into the model-based procedur e. This strategy has led to the structural determination of two viruses tha t, ill our hands, resisted solution by other means.