Fig. 3.

Overview of steps for the determination of single-particle cryo-EM 3D reconstructions. Cryo-EM images are acquired and individual particles are extracted manually or with automation, ideally followed by user examination to exclude falsely picked projections. The particle projections are subjected to 2D classification and averaging, aiming to group the projections according to their similarity. An initial 3D model may be generated ab initio using class averages representing different particle orientations, by geometrically constrained 3D reconstruction methods such as by RCT, or by utilizing available structural information. The initial 3D model can serve as a reference for 3D classification of the data, a useful step for identifying distinct conformations or macromolecular compositions. The 3D classification also facilitates identification of particle subsets that are amenable to higher resolution refinement. Depending on the achieved resolution, the final maps can be used for de novo structure building or the modeling of available structures based on various criteria.