Award

Limitations in sample preparation have become increasingly apparent as a significant limitation to the throughput, resolution, and accuracy of micrographs in cryo-EM. Technological advances in cryo-EM have increased the resolution achievable, and in turn, have revealed the extent to which the air-water interface (AWI) damages biomolecular structures. The AWI is the boundary between the aqueous sample surface and the surrounding environment, before vitrification. The AWI causes preferential orientation of sample molecules and damage to the tertiary structure, preventing accurate 3D reconstructions of the native states of biological structures. The streptavidin affinity grids that will be developed in this proposal are used to tether samples to the grid surface and prevent interactions with the AWI, resulting in a higher percentage of samples suitable for high-resolution imaging per sample grid, and subsequently, higher throughput. The surface of the streptavidin affinity grid is spanned by monolayer crystals of streptavidin, providing support for biotinylated sample molecules and preventing travel to the AWI before vitrification. This preparation technique prevents sample damage or the adoption of preferred orientations. In collaboration with Lawrence Berkeley National Laboratory, where these grids were originally developed, Phase I of this STTR project will demonstrate the feasibility of batch production of the streptavidin affinity grids. The work plan will include prototyping and development of fixtures to process multiple grids at once using the preparation procedure for single grids developed by Dr. Han at LBNL. Additionally, grids will be tested to validate quality, uniformity, and repeatability in the batch preparation process before Phase II manufacturing development and automation of the procedure. Commercial availability of these grids will provide a widely applicable solution to the AWI for cryo- EM laboratories and remove a significant barrier to the throughput of high-resolution 3D reconstructions of biological sample structures. Structural research provides the basis for further research like predicted new bioenergy materials, increasing the efficiency of biomanufacturing, or developing synthetic alternatives. Overall, these improvements are essential to the movement towards cleaner, more efficient energy.