The technology development cores in our parent grant include the near atomic resolution structural determination of large molecular machines, single particle averaging of subtomograms in cryo-ET of complex assemblies and cells, data integration from the wet lab through 3-D reconstruction and deposition, and exploring the technique of Zenike phase contrast optics. This supplement focuses primarily on Zernike phase contrast imaging, which among all of our cores, shows the greatest potential for revolutionary change in the field. With other new resource, we are also installing a direct electron detection device which has the potential, for the first time, to surpass the quantum efficiency of photographic film. For traditional single particle imaging, per-image efficiency does not fundamentally limit what can be achieved. However, for the applications where Zernike phase contrast is the most effective, per-image efficiency is a major factor. Combining these two technologies, with the addition of an in-column energy filter, offers the potential for tackling previously impossible structural projects at higher resolutions. However, these technologies are far from maturity, and in this supplement, we work to resolve some of the current limiting factors in these technologies.
Our aims are: 1) Assessing the limiting factors contributing to the short lifetime of the current Zernike phase plate. 2) Fabricating and testing Zernike phase plates with improved lifetime and high resolution imaging capabilities using the clean room nanofabrication facility at Caltech. 3) Testing the performance specifications of the new direct electron detection device for high quality image collection. 4)Developing new image processing protocols for optimally working with Zernike images recorded on the direct detection device. This technology development is driven by recently established collaborative structural projects including (i) biochemically purified RNA molecules as small as 50 kDa, which are generally considered to be difficult or impossible due to their small size;and (ii) phages at different stages of infection and assembly. These specimens are highly relevant to cancer therapeutics and diagnostics and to viral infection.
In this supplement proposal, we would like to extend the capability of cryo-EM and cryo-ET in solving structures of (i) biochemically purified biological molecules ranging from 50-400 kDa;(ii) bacteriopahges at different stages of infection and assembly. These specimens are highly relevant to cancer therapeutics and diagnostics and to viral diseases.
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