Determining the structure of biological macromolecules by transmission electron microscopy, single particle analysis and 3D reconstruction

Single particle analysis and 3D reconstruction of molecules imaged by trans mission electron microscopy have provided a wealth of medium to low resolut ion structures of biological molecules and macromolecular complexes, such a s the ribosome, viruses, molecular chaperones and photosystem II. In this r eview, the principles of these techniques are introduced in a non-mathemati cal way, and single particle analysis is compared to other methods used for structural studies. In particular, the recent X-ray structures of the ribo some and of ribosomal subunits allow a critical comparison of single partic le analysis and X-ray crystallography. This has emphasised the rapidity wit h which single particle analysis can produce medium resolution structures o f complexes that are difficult to crystallise. Once crystals are available. X-ray crystallography can produce structures at a much higher resolution. The great similarities now seen between the structures obtained by the two techniques reinforce confidence in the use of single particle analysis and 3D reconstruction, and show that for electron cryo-microscopy structure dis tortion during sample preparation and imaging has not been a significant pr oblem, The ability to analyse conformational flexibility and the ease with which time-resolved studies can be performed are significant advantages for single particle analysis. Future improvements in single particle analysis and electron microscopy should increase the attainable resolution, Combinin g single particle analysis of macromolecular complexes and electron tomogra phy of subcellular structures with high-resolution X-ray structures may ena ble us to realise the ultimate dream of structural biology a complete descr iption of the macromolecular complexes of the cell in their different funct ional states, (C) 2001 Elsevier Science Ltd, All rights reserved.