Protocols for Subtomogram Averaging of Membrane Proteins in the Dynamo Software Package

Cryo-electron tomography allows low-resolution three-dimensional (3D) viewing of cellular organelles and macromolecular complexes present as multiple copies within a tomogram. These structures are computationally extracted and averaged in order to obtain high-resolution 3D structures, and provide a map of their spatial distribution and interaction with their biological microenvironment. To do so, we apply the user-friendly Dynamo software package on a tomographic data set. Dynamo acts as a modular toolbox adaptable to different biological scenarios, allowing a custom designed pipeline for subtomogram averaging. Here, we use as a textbook example the mitochondrial docking site of the positive-strand RNA Flock house nodavirus (FHV) to describe how Dynamo coordinates several basic steps in the subtomogram averaging workflow. Our framework covers specific strategies to deal with additional issues in subtomogram averaging as tomographic data management, 3D surface visualization, automatic assignment of asymmetry and inherent loss of Fourier information.