Abstract
Cryo-electron microscopy and single-particle image analysis are frequently used methods for macromolecular structure determination. Conventional single-particle analysis, however, usually takes advantage of inherent sample symmetries which assist in the calculation of the structure of interest (such as viruses). Many viruses assemble an icosahedral capsid and often icosahedral symmetry is applied during structure determination. Symmetry imposition, however, results in the loss of asymmetric features of the virus. Here, we provide a brief overview of the methods used to investigate non-symmetric capsid features. These include the recently developed focussed classification as well as more conventional methods which simply do not impose any symmetry. Asymmetric single-particle image analysis can reveal novel aspects of virus structure. For example, the VP4 capsid spike of rotavirus is only present at partial occupancy, the bacteriophage MS2 capsid contains a single copy of a maturation protein and some viruses also encode portals or portal-like assemblies for the packaging and/or release of their genome upon infection. Advances in single-particle image reconstruction methods now permit novel discoveries from previous single-particle data sets which are expanding our understanding of fundamental aspects of virus biology such as viral entry and egress.