Saturday, September 7, 2024
9/7/2024
Project summary Cryogenic electron microscopy (cryo-EM) has recently emerged as a powerful tool to obtain high- resolution protein structures in their native state. Although major advances in hardware, data collection, and data processing have enabled close to angstrom resolution of protein structures, obtaining good specimens remains challenging and has become the bottleneck in single-particle cryo-EM. Cryo-EM requires protein molecules embedded in a very thin layer of vitreous ice. This is not easy because some proteins diffuse quickly to the air-water interface of such a thin sample before vitrification and immediately undergo denaturation, aggregation, or adopting preferred orientations. These events render the sample unsuitable for cryo-EM. Also, problems with protein particles not going into the holes of the sample grid are encountered. We hypothesize that protein particles can be distributed more uniformly on and maintain proximity to the grid surface by covalent attachment. This proposal seeks to facilitate quality specimen preparation by developing a specimen platform that utilizes the tetrazine ligation, a highly efficient biorthogonal reaction, to capture and covalently attach protein particles to the grid surface. We will achieve this objective by pursuing two specific aims: 1) develop tetrazine functionalized monolayer graphene grids, and 2) evaluate the quality of the resulting grids for single-particle cryo-EM. Unlike affinity grids that rely on reversible noncovalent interactions to bring proteins close to the grid surface, our covalent capture approach is innovative in capturaing proteins irreversibly. This prevents the release of already bound particles and is particularly advantageous for low protein sample concentrations.
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