ANGULAR RECONSTITUTION IN THREE-DIMENSIONAL ELECTRON MICROSCOPY: PRACTICAL AND TECHNICAL ASPECTS

Cryo-electron microscopy in combination with the angular reconstitution technique has become an important and rapid technique for analyzing the three-dimensional (3D) structure of individual, non-crystallized biological macromolecules. Behind this technique is the idea that a 3D structure can be calculated based on a very large number of images of individual molecules randomly oriented within a homogeneous matrix. The approach has great practical and theoretical advantages over earlier techniques which require tilting of the specimen holder in the electron microscope. We want to elaborate on some of the specific details of our procedures. For example, alignments are used to remove the “in-plane” positional x- and y-shifts, as well as the inplane rotations of the molecules. Automatic classification procedures are subsequently used to find the different characteristic views of the molecules due to “out-of-plane” rotations of the molecules. The characteristic views are assigned Euler angles by the angular reconstitution technique using sinograms and sinogram correlation functions under the boundary conditions dictated by the pointgroup symmetry of the structure. Three-dimensional reconstructions can thus be calculated; and once a preliminary reconstruction is available, iterative refinements are applied to further improve the quality of the results.