Subtomogram averaging of helical filaments in cryo-electron tomography

Summary Helical filaments are essential macromolecular elements in cellular organization and dynamics. Recent advances in cryo-electron tomography allow faithful imaging of isolated or in-cell helical filaments. Here, we present a protocol to generate density maps at sub-nanometer resolution of helical filaments by subtomogram averaging, exemplified with isolated mumps virus nucleocapsids and their in-cell form as an extension of the method. We detail procedures from pre-processing of tilt series frames to refinement of reconstructed averages for streamlined data processing of helical filaments.