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bioRxiv · 10.1101/2025.08.13.669967

Cryo-EM protein structure without purification

Powell, S.·Evans, J. E.
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Abstract

Cryo-electron microscopy (cryo-EM) is a powerful technique for macromolecular structure determination. While several aspects of the workflow are either semi- or fully automated, the beginning of the workflow related to the generation of macromolecular samples remains a bottleneck limiting full automation. In most cases, the production of the initial sample still takes days to months. Initial sample production often takes days to months. Here, we show that cryo-EM can solve structures of proteins directly from cell-free expression complex mixtures with minimal or no purification while also reducing reagent volumes and overall labor and time. Using a cell-free reaction volume of just 25 microliters, we achieve rapid protein synthesis and structural characterization in under 24 hours at resolutions better than 3[A]. These results highlight a lower cost and rapid gene to structure workflow with minimal perturbation satisfies the foundational requirements needed to facilitate future improvements in throughput via full workflow automation.

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Powell, S., Evans, J. E.. 2025-08-13. Cryo-EM protein structure without purification. https://doi.org/10.1101/2025.08.13.669967

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