3-D Structures of Macromolecules Using Single-Particle Analysis in EMAN

Single-particle reconstruction is a methodology whereby transmission electron microscopy (TEM) is used to record images of individual monodisperse molecules or macromolecular assemblies, then sets of images of individual particles are computationally combined to produce a 3-D volumetric reconstruction. Ideally the TEM specimen will be prepared in vitreous ice (electron cryomicroscopy), but negative stain preparations may be used for lower resolution work. This technique has been demonstrated to produce structures at resolutions as high as ∼4 �, though this is not yet typical. The reconstruction process is quite computationally intensive, and several software packages are available for this task. EMAN is one of the easier to master software suites for single-particle analysis. This protocol explains how to perform an initial low-resolution reconstruction using EMAN.