3D Variability Analysis: Directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images

Proteins are dynamic molecules that constitute the molecular machinery of living cells. Single particle cryo-EM excels as an imaging technique to solve static structures of proteins, but existing computational 3D reconstruction methods for cryo-EM data have not been effective in practice for solving structures or modelling motion of flexible proteins. We introduce 3D variability analysis (3DVA), an algorithm that models the conformational landscape of a molecule as a linear subspace, and is able to fit the model to experimental cryo-EM data at high resolution. 3DVA makes it possible, for the first time, to resolve and visualize detailed molecular motions of both large and small proteins. Through extensive experimental results on cryo-EM data, we demonstrate the ability of 3DVA to resolve multiple flexible motions of α-helices in the sub-50 kDa transmembrane domain of a GPCR complex, bending modes of a sodium ion channel, five types of symmetric and symmetry-breaking flexibility in a proteasome, and large motions in a spliceosome complex. We also show that 3DVA can uncover discrete conformational states of heterogenous mixtures of ribosome particles from a single sample. The results demonstrate that 3DVA enables new biological insight to be extracted from single particle cryo-EM data. 3DVA is implemented in the cryoSPARC software package, and has already been used successfully in several notable structural studies.