With the resolution revolution in cryo-EM came an influx of high-resolution structures of members of the TRP channel family, which had not been amenable to structural characterization via X-ray crystallography. A handful of the members of this ion channel family have a peculiar temperature sensing property, but knowing their structures has yet to shed light on the molecular and thermodynamic basis of this property. With this motivation in mind, here we will focus on outstanding problems in cryo-EM that contribute to the difficulty of understanding these biological macromolecules. This includes topics ranging from the use of quaternions for estimating the orientation of 3D particles from 2D images within a machine learning context to a discussion of methods for resolving continuous heterogeneity in single-particle cryo-EM. Overall, while our motivation stems from understanding a particular biological system, the issues discussed here are widely relevant to cryo-EM and cryo-ET as a whole.
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