Single-particle analysis of electron cryo-microscopy (cryo-EM) images allows structure determination of macromolecular complexes. But when these molecules adopt many different conformations, traditional image processing approaches often lead to blurred reconstructions. By considering complexes to be comprised of multiple, independently moving rigid bodies, multi-body refinement in RELION enables structure determination of highly flexible complexes, while at the same time providing a characterization of the motions in the complex. Here, we describe how to perform multi-body refinement in RELION using a publicly available example. We outline how to prepare the necessary files, how to run the actual multi-body calculation, and how to interpret its output. This method can be applied to any cryo-EM data set of flexible complexes that can be divided into two or more bodies, each with a minimum molecular weight of 100-150 kDa.
Keywords: Cryo-EM; Image classification; Molecular motions; RELION; Regularized likelihood optimization; Single-particle analysis; Structural biology.