Processing of Transmission Electron Microscopy Images for Single-Particle Analysis of Macromolecular Complexes

International audienceThree-dimensional (3D) structure of a wide range of biological macromolecular assemblies can be computed from two-dimensional images collected by transmission electron microscopy. This information integrated with other structural data (e.g., from X-ray crystallography or nuclear magnetic resonance) helps structural biologists understand the function of macromolecular complexes. Single-particle analysis (SPA) is a method used for studies of complexes whose structure and dynamics can be analyzed in isolation. To reconstruct the 3D structure, SPA methods use a large number of images of randomly oriented individual complexes. When the angular distribution of single-particle orientations samples Fourier space completely and the population is structurally homogeneous, a resolution of the reconstruction of 0.4-1 nm can be achieved. Such high resolutions are possible thanks to the high number of images and the correction of the Contrast Transfer Function (CTF) of the microscope. One of the standard SPA approaches is the refinement of a preliminary 3D model using iterative projection matching combined with CTF correction. We describe a protocol for the refinement of a preliminary model using CTF correction by Wiener filtering of volumes from focal series of experimental images. This protocol combines potentially best features of two other protocols proposed in the field