Angular reconstitution of ice embedded macromolecules with arbitrary point group symmetry

Abstract

Electron microscopy of individual non-crystallized (large) macromolecules is a very rapid technique for probing the three-dimensional (3D) structure of biological macromolecules. Since there is no need for extensive crystallization experiments, specimen preparation can be simple and fast. In particular the vitreous-ice embedding specimen preparation technique, in which the macromolecules are kept in a waterlike environment, has proved very suited for this purpose. One may extract three-dimensional information from the data - without ever collecting tilt series in the microscope - by exploiting thedifferent ("random") orientations the macromolecules have with respect to the grid. Collecting tilt series can be cumbersome and requires multiple exposure of the sensitive molecules. The only techniquewhich was available for such single-shot 3D microscopy was the common-lines technique for virusses with icosahedral symmetry [Crowther (1971), Fuller (1987)]. The other technique available for asymmetric non-crystalline specimens is the random conical tilt technique [Radermacher (1986)]. However, this technique is a tilt series technique requiring two exposures per specimen area. Moreover, it requiresthe molecules to be preferentially oriented with respect to the plane of the support film which - in turn - requires strong (and thus unfavourable) interactions between molecule and support.