Sunday, April 28, 2024
4/28/2024
PROJECT SUMMARY/ABSTRACT High-resolution electron cryo-microscopy (cryo-EM) has recently become a routinely-used technology within biochemistry, cell biology, and structural biology, fields that are essential to the fundamental understanding of wellness and the treatment of disease. Use of high-resolution cryo-EM can become even more wide-spread and productive than it is at present, if the step of grid preparation is made to be much more reliable. To that end, we here propose to explore the development of a new technology for consistently making large areas of EM grids that have the right thickness desired for high-resolution data collection. The approach that we will explore is to blot only from the perimeter of grids, using – for example – filter paper with a 2 mm hole that is centered on the 3 mm EM grid. This perhaps-counterintuitive approach differs greatly from the current practice of pressing filter paper over the entire face of a grid. While the underlying principles of physics, which govern the flow of excess water from the grid into the filter paper, are the same in the two situations, the boundary conditions – describing where liquid bridges exist between the surface of the EM grid and the filter paper – are very different, The concept of draining liquid from the perimeter is traceable to, and draws insight from, early studies in the physical chemistry of thin liquid films, in which that geometry was the natural choice, allowing interference microscopy to be used to observe the formation and stability of such films. If draining from the perimeter now proves to be successful in reliably making thin liquid films on EM grids, the technology of “blotting with a hole” should significantly accelerate what is accomplished by single-particle cryo-EM.
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