Image analysis of single macromolecules

A battery of sophisticated techniques is now available to extract three-dimensional structural information from electron micrographs of biological macromolecules occurring in the form of single particles. One of these techniques, the random-conical reconstruction method, which allows low-dose imaging, has been recently perfected and is being used routinely for the study of ribosomal architecture. The analysis of the 40S mammalian ribosomal subunit serves as an illustration of the various steps of image processing. The use of classification combined with 3-D reconstruction provides the means to investigate variations of the macromolecular structure (deformations, conformational changes, etc.) that are caused by the specimen preparation. An example is provided by the changes in the shape of the 70S monosome of E. coli as it changes its orientation on the carbon grid. The most challenging applications of the techniques discussed are in the area of cryo-microscopy of ice-embedded specimens. First studies of single macromolecules imaged in this way have indicated that the 3-D imaging methods and, specifically, the random-conical reconstruction method, will be applicable under these conditions.