ABSTRACT
The recent advent of three-dimensional (3-D) image reconstruction methods in electron microscopy (EM) that permit the determination of the structure of virtually any biological macromolecule or macromolecular complex, isolated or in regular arrays, has brought about a renaissance in which electron microscopy plays a more prominent role than ever before in the study of biological structure and function. Historically, a biologist wishing to pursue electron microscopy and image analysis had been limited to interpreting two-dimensional (2-D) projection images of objects, whereas it was their 3-D structures that were intimately related to their function. Only in special cases, when biological specimens had high internal symmetry, such as icosahedral viruses or helical particles, and when the specimen could be experimentally coaxed to crystallize in two-or three-dimensional arrays or fortuitously exhibited preferred orientations, could 3-D structures be determined. It is now possible to add a final and general approach to these techniques to study the 3-D structure of any pure biological macromolecule or macromolecular complex with a method known as quaternion-assisted angular reconstitution.1-4 This method requires several hundred to a few thousand images of the biological macromolecule of interest, depending on the desired level of detail, and consequently demands only small amounts of purified biological substance. It uses images of sets of single particles of the biological macromolecule at random orientations; crystals or preferred orientations are not required. The approach can be and has been applied to both bright-field images of stained macromolecules and to images of biological particles cryo-prepared and imaged unstained in vitreous ice. It can be and has been applied to dark-field scanning transmission electron microscope images of stained particles or unstained freeze-dried proteins,4•5 and to microanalytical energy-filtered images of macromolecules and macromolecular complexes.6•7 Additionally, in comparison to other approaches in structural biology such as x-ray crystallography, NMR spectroscopy, and even electron microscopy-based crystallography, the required time for analysis by the quaternion-assisted angular reconstitution approach is significantly shorter. The following sections first introduce in more detail the different types of electron microscopy that have been used with the quaternion-assisted three-dimensional image reconstruction procedure. Advances in the quaternionassisted angular reconstitution method are subsequently presented and discussed, with a brief comparison to other electron microscopic determination methods. Examples of the quaternionassisted reconstitution method are also given in the context of its application to biological questions related to: the storage of genetic information, the expression of genetic information through the process of transcription, the synthesis of proteins, and protein trafficking to appropriate cellular compartments. These examples also demonstrate how the use of quaternion-assisted angular reconstitution has shed light on the functions and related conformational changes of macromolecular complexes that have eluded structural analysis using other methods.
