This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACT:A serious limitation of the microdroplet spraying with on-grid mixing of components and flash photolysis techniques is that the reactions take place on the surface of a carbon-coated grid, and it seems questionable whether reactions involving macromolecular complexes will occur in this environment in the same way as they do in the test tube. Using holey grids could help, but this is not always feasible and does not eliminate interactions of the reactants with the carbon film or at the air-water interface that is present prior to freezing. The pre-mix mode is preferred, but since it relies on blotting with filter paper following deposition of the reaction mixture on the grid, the time resolution is poor (i.e., in the order of seconds). Ideally, kinetics experiments should be conducted in bulk phase in vitro under conditions that are as close to physiological as possible, and at selected time intervals samples should be taken and frozen on EM grids instantaneously. The micro-mixer/sprayer described in the previous section is one approach to solving these problems. In another approach, we are working to assess the feasibility of micro/nanofabrication and micro- and nanofluidics technologies for performing fast (millisecond time-scale) pre-mix modes of operation by demonstration of a functioning microfabricated prototype device that mixes sub-microliter volumes of reactants either directly on a modified grid or before application to the grid, and then freezes the grid near-instantaneously. Successful implementation would establish this as the method of choice for time-resolved cryo-EM of actively functioning macromolecular assemblies, and would lay the groundwork for more sophisticated devices.
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