This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This core project is aimed at setting up an experimental and computational procedure to address structural questions by using electron cryo-tomography (cryo-ET) followed by post- tomographic classification, alignment and averaging. Cryo-ET has emerged in recent years as a powerful technique for bridging the gap between atomistic structures and whole cells. Specifically, the recent idea to perform so called 'single particle' cryo-ET is closely related to the existing expertise at the NCMI, and we believe we can make a contribution in this area by leveraging our experience with high resolution data collection and processing. Tomography can help solve the problem of multiple conformational states posed above because of its unique ability to give three-dimensional information about a macromolecular assembly, greatly reducing the confusing interaction of structural heterogeneity with orientation that is often difficult to distinguish with a single 2D image of each particle.