During the past few years analog and digital image processing techniques have become indispensable tools for the study of protein structure by electron microscopy [for reviews see e.g. 1,2,3]. The two most frequently used among these techniques are ‘image enhancement’ and ‘structure reconstruction'. Image enhancement techniques are used to objectively extract the reproducible structural features contained in noisy images recorded from the specimen under investigation, by combining their redundant information by means of ‘real space averaging’ or ‘Fourier space filtering'. The prerequisite for the ultimate success of these techniques is the ability to properly align the images relative to each other. The purpose of structure reconstruction techniques is to determine the three-dimensional structure of proteins by combining a set of sufficiently independent two-dimensional projection images recorded from the object under study - hence three-dimensional reconstruction techniques. This step becomes necessary, because biological specimens are generally translucent to the electron illumination, and therefore micrographs recorded from them in transmission mode represent projection images of the inherently threedimensional specimen that superimpose structural features from different depth within it.
Read full abstract