Subtomogram averaging for biophysical analysis and supramolecular context

Recent advances in hardware, software and computing power have led to increasingly ambitious applications of cryo-electron tomography and subtomogram averaging. It is now possible to reveal both structures and biophysical relationships like protein binding partners and small molecule occupancy in these experiments. However, some data processing choices require the user to prioritize structure or biophysical context. Here, we present a modified subtomogram averaging approach that preserves both capabilities. By increasing the accuracy of particle-picking, performing alignment and averaging on all subtomograms, and decreasing reliance on symmetry and tight masks, the usability of tomography and subtomogram averaging data for biophysical analyses is greatly increased without negatively impacting structural refinements.