1a Maximizing information in highly complex cryo-EM data

Cryo-electron microscopy single particle reconstruction (cryo-EM SPR) provides detailed structural information for macromolecules by averaging many images of individual molecules. However, accurate image alignment is a bottleneck due to the poor contrast of individual molecules images. Therefore, in practice, structures of only relatively large macromolecules can be determined by cryo-EM SPR. Phase plates are devices that enhance the signal in cryo-EM images, but current approaches to data collection do not allow us to capitalize on this enhancement. In order to achieve dramatically higher contrast, a much more precise data collection setup is required. The overall objective of this Phase II SBIR proposal is to develop enabling technologies for optimal use of the Volta phase plate so its contrast improvement potential can be realized. For practical reasons, this requires ultra-fast analysis of huge image datasets in real-time, necessitating the development of GPU-based algorithms. These methods will enable highly efficient data collection with the high level of precision necessary to use phase plates close-to-focus where the contrast enhancement is the highest. We will develop, implement, and test multiple calibration procedures, with the faster ones performed for each data collection area, while slower, more elaborate ones are performed much less frequently. We will implement full data collection control with real-time data analysis to provide feedback and initial approximation to final results. The redesign of the approach to data collection using a phase plate will benefit all types of cryo-EM SPR and fiber reconstruction: (1) the resolution of many reconstructions will improve significantly, (2) cryo-EM will be applicable to projects where particles have lower mass, and (3) molecular motions for larger particles can be better identified and analyzed. These benefits are of high significance for this quickly expanding field, so the potential for successful commercialization is high.