Many of the electron microscopy (EM) samples that are analyzed by single-particle reconstruction are flexible macromolecular assemblies that adopt multiple structural states in their functioning. Consequently, EM samples often contain a mixture of different structural states. This structural variability has long been regarded as a severe hindrance for single-particle analysis because the combination of projections from different structures into a single reconstruction may cause severe artifacts. This chapter reviews recent developments in image processing that may turn structural variability from an obstacle into an advantage. Modern algorithms now allow classifying projection images according to their underlying three-dimensional (3D) structures, so that multiple reconstructions may be obtained from a single data set. This places 3D-EM in a unique position to study the intricate dynamics of functioning molecular assemblies.
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