Tutorial: Single Particle Analysis of Macromolecules and Complexes: How to Get Started

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Single particle processing is a technique for determining the 3D structure of molecules and macromolecular assemblies using TEM images of individual molecules. In principle, individual molecules float freely in solution before freezing/fixation, so particles in the micrographs represent 2D projections of individual molecules in random, or nearly random orientations. Practically speaking, surface effects often cause preferred orientations to exist in the particle population. In addition, single particle processing makes the assumption that all of the individual molecules are either in the same conformation, or in a discrete mixture of conformations. Single particle processing refers to the computational techniques by which thousands of individual molecule images are processed to produce a 3D structure. The technology of single particle processing has advanced to the point that a low resolution reconstruction (∼ 15-25 Å) can be performed on a cooperative specimen quite rapidly, typically less than a month from purified specimen to 3D model.