Because isolated macromolecules, prepared on a carbon support film or in a thin layer of ice over a holey carbon film, usually exhibit a full range of orientations, resulting projections will differ as well, and substantial processing is needed before averaging can take place. Basically, the method of single particle analysis consists of only a few crucial steps, of which two are illustrated in Fig. 2. If projections result from one type of orientation on the support film, GSK872 cell line averaging is possible after alignment. The alignment step brings projections in equivalent positions by computing rotational

and translational shifts. In the case of the example, a supercomplex of trimeric photosystem I (PSI) surrounded by a ring of 18 copies of the antenna

protein IsiA, a set 17DMAG of 5000 projections has been brought in register. It can be seen that by increasing the number of summed projections the noise is gradually reduced (Fig. 2, upper part). It is very obvious that from individual, noisy projections the number of IsiA copies cannot be retrieved and that processing is indispensable. Fig. 2 The basics of single particle EM, explained from an analysis of the photosystem I–IsiA supercomplex from the cyanobacterium Synechococcus 7942, extracted from ACY-241 research buy negatively stained EM specimens (Boekema et al. 2001). After translational and rotational alignment of a data set of about 5000 single particle projections showing the complex in a position as in the membrane plane, sums with increasing numbers of copies in equivalent

positions show the gradual improvement in the signal-to-noise ratio (upper part of the picture). However, these particle projections may not all be identical, because small tilt variations on the membrane plane may lead to different positions. Indeed, after multivariate statistical analysis and classification, it became clear that only a small number of projections show threefold rotational symmetry which is indicative for a position parallel to the membrane (lower row, left). The other two classes Demeclocycline (middle and right) show the supercomplex in tilted positions Just summing of projections, however, is meaningless when the projections arise from particles in different orientations toward the plane. In order to deal with this, data sets have to be treated with multivariate statistical analysis together with automated classification (see Van Heel et al. 2000; Frank 2002 for reviews on single particle EM). After statistical analysis and classification, those images that are most similar can be grouped together. The output of the classification is “classes” of groups of homogeneous projections.