Electron crystallography has been used for decades to determine three-dimensional structures of membrane proteins embedded in a lipid bilayer. However, high-resolution information could only be retrieved from samples where the 2D crystals were well ordered and perfectly flat. This is rarely the case in practice. We implemented in the FOCUS package a module to export transmission electron microscopy images of 2D crystals for 3D reconstruction by single particle algorithms. This approach allows for correcting local distortions of the 2D crystals, yielding much higher resolution reconstructions than otherwise expected from the observable diffraction spots. In addition, the single particle framework enables classification of heterogeneous structures coexisting within the 2D crystals. We provide here a detailed guide on single particle analysis of 2D crystal data based on the FOCUS and FREALIGN packages.