New methods for characterising heterogeneous populations of macromolecules using electron microscopy-defining function by direct imaging of states

A goal of life scientists is to understand how the molecules in cells operate and interact. We have a good understanding of isolated protein domains, and we can see the overall organisation of cell structure with light and electron microscopy. The resolution obtained with techniques in molecular microscopy bridges the gap between the high resolution information on isolated domains, and the lower resolution overview given by light microscopy and the conventional electron microscopy techniques. It is at the intermediate scale that the molecules are directly involved in regulating cellular processes. Biological macromolecular complexes are often molecular machines in that they consist of many parts with distinct functions, and movements or conformation changes are integral to their function and operation. Through observation of these complexes in their different states we could gain significantly improved understanding and insight on how these complexes work and function in the cell. Many fundamental cellular processes, such as DNA transcription, translation and repair; or protein synthesis, chaperoning, transport and degradation, are mediated by such large multi-component macromolecular complexes. With recent advances in electron microscopy instrumentation and analysis techniques we can examine the structures of these complexes in three dimensions at sub nanometre resolution. However the presence of mixed conformations can be a major limiting factor for this technique, as usually the assumption that all the molecules or particles are identical is applied. If a mixture of conformations or structures is present, and these are not detected and separated, then they will be averaged and the obtainable resolution will be limited. The challenge is that if the conformation species could be separated then invaluable information on function, dynamics, and mechanism could be obtained In this project we will develop a technique that will allow all the discrete states in the molecular population to be analysed, making the presence of heterogeneity an advantage, as the observation of multiple states reveals much about the function and mechanism in macromolecular complexes. The novel method provides an objective analysis of a mixed population of particles, from electron micrographs. It enables images of the states to be separated, so that each homogeneous class can be processed by the standard methods. Until now sample heterogeneity has been the biggest limiting factor for single particle analysis, but once this problem is solved it will become an advantage. Understanding the molecules involved in cellular processes enhances our knowledge of the mechanism of life, and provides a rational basis for the treatment of diseases.

Single particle analysis by electron microscopy is a powerful and versatile technique in structural biology, and near atomic resolutions have been be reached with some specimens. The main limitations in single particle analysis are specimen dependent, and one of the main limiting factors is heterogeneity in the preparation of molecules imaged. Large macromolecular complexes mediate many fundamental cellular processes, such as DNA transcription, translation and repair; or protein synthesis, chaperoning, transport and degradation. Through observation of these complexes in their different states using electron microscopy we can gain a significant step-up in our levels of understanding and insight on how these complexes work and function in the cell. The 2D images recorded in transmission electron microscopy are projections of the 3D molecular structures. The problem is that in projections of the structures, views of different orientations can not be distinguished from conformation changes or views of other molecules. In fact small conformation changes will have more subtle effects on the projections than large orientation differences. If these different images are mixed incorrectly, then the structure determined will be poor. The challenge is that if the conformations or species could be separated then invaluable information on function, dynamics, and mechanism could be revealed. This novel methodology will survey the molecular population based only on electron micrograph images collected, with no prior or assumed information of the structure/s. The number of discrete classes and the level of population in the discrete states will be indicated. Then the images will be separated into clusters belonging to each of the discrete states, and their structures computed by standard single particle methods. Currently sample heterogeneity is one of the biggest limiting factors for single particle analysis, but once this problem is solved it will become a strength.

Impact plan This is an enabling technology, meaning it is not directly a product or service, but it could enable the creation of these. By making it as widely available as possible it will have the maximum impact, and this may enable new strategies and applications that would not have been possible before. The full extent of these is unpredictable. The strategy is for a wide distribution and too make it freely available. It will beadvertised inor research publications. Software is easy to distribute, so it is realistic to aim for a wide uptake, There could be three levels of impact: 1. Direct user of technology. These will get use the capabilities to produce new results. a. New capabilities b. Efficiency savings 2. Those who use the results provided by the direct users. This includes anyone who makes a decision based on that information, be it to guide an new line of investigation, or avoid one. Also to develop new products or therapies. 3. The 3rd level will be the users of products or therapies that have benefitted from the new enabling technology. Because the line to products is indirect, it is difficult to estimate any level of financial figure for the impact to the UK economy. However increased efficiency in research labs or clinical departments will be a direct impact. It is possible that it will lead to clinical or screening applications in the health service or industry.