IMPROVEMENT OF TIME-RESOLVED INSTRUMENTATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT The goal of time-resolved cryo-electron microscopy studies is to characterize the structures of intermediates that arise during the functioning of macromolecules. Developments in instrumentation, specimen preparation, and data analysis are carried out using specimens from existing in-house projects that benefit from time-resolved imaging: intermediate states of translating ribosomes, functional states (open, closed, inactivated/adapted) of the ryanodine receptor class of calcium release channel, and metabolic states of the mitochondrion. For the large complex assemblies of macromolecules that are of interest here, some of these intermediates are expected to involve movements of entire domains or subunits, and loss or gain of component subunits (e.g., as in the assembly or disassembly of complexes) or macromolecular ligands (e.g., tRNAs interfacing with the ribosome). These types of structural changes should be detectable at resolutions that are typically attained by the single-particle cryo-EM approach. The lifetime of the intermediates must be in the millisecond range or greater to be accessible with current technology, and there are indeed many examples where this is known to be the case. This project is a continuation of our efforts to implement and refine existing time-resolved modalities so that intramural and extramural collaborative projects can commence efficiently and productively. Specific aim 2 represents an entirely new innovative project that will be done in close collaboration involving a contractual agreement with Dr. Toh-Ming Lu and his associates of the Center for Integrated Electronics (CIE) at the nearby Rensselaer Polytechnic Institute. A Wadsworth Center scientist with expertise in microfabrication, Dr. Hisham Mohamed is also contributing to these efforts.