A number of cellular processes are carried out by large assemblies constituted by a well-defined set of protein components. These large assemblies can be considered as "macromolecular machines", in which individual proteins act as parts of the machine that make specific contributions to its function. The characterization of such large complexes constitutes the next frontier in structural biology research. Structural characterization of large macromolecular complexes is best carried out using a combination of techniques. High resolution structures of individual components are placed in lower resolution maps of the entire complex. Characterizing different conformations of the complex can significantly help to establish its mechanism of action.
Macromolecular Electron Microscopy
The sensitivity of biological molecules to radiation damage limits the amount of information that can be obtained when some type of radiation is used to interrogate their structure. Crystallographic techniques overcome this signal-to-noise limitation by obtaining information from very large numbers of identical molecules, all arranged in a particular conformation in a crystal. Although techniques such as X-ray crystallography can provide near-atomic resolution information, their application depends on being able to obtain crystalline arrays (a process that often requires large amounts of very pure material), and on being able to derive phase information for the diffraction data. Furthermore, the forces that result in formation of a crystal affect the may affect the conformation of a molecule, and result in an environment that can differ significantly from that in which the molecule naturally functions.
The technique of macromolecular single-particle electron microscopy relies on the observation that a suitably recorded electron microscope image of a single large (MW > 250,000Da) molecule contains enough information to accurately determine the orientation of the molecule. Therefore, starting with a very small amount of material, information from many such molecules can be computationally combined, providing an alternative way to overcome signal-to-noise limitations, and obtain structures at intermediate (10-25Å) resolution without the use of crystalline arrays. The images contain all information required (no phase problem to deal with), and are captured under nearly physiological conditions, which may provide valuable information about the functionally relevant quaternary structure of the system.