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bioRxiv · 10.1101/2025.01.10.632437

MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

Eluru, G.·De Gieter, S.·Schenck, S.·Stroobants, A.·Shrestha, B.·Erbel, P.·Brunner, J. D.·Efremov, R. G.
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Abstract

Single particle cryo-EM enables reconstructing near-atomic or even atomic resolution 3D maps of proteins by visualizing thousands to a few million purified protein particles embedded in nanometer- thick vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of one of the most sensitive analytical methods that deliver a high-resolution protein structure as a readout. In practice, more than a million times more starting biological material is required to prepare cryo-EM grids. To close the gap, we developed a micro isolation (MISO) method that combines microfluidics-based protein purification with cryo-EM grid preparation. We validated the method on soluble bacterial and eukaryotic membrane proteins. We showed that MISO enables protein structure determination starting from below one microgram of a target protein and going from cells to cryo-EM grids within a few hours. This scales down the purification by a factor of a few hundred to a few thousand and opens possibilities for the structural characterization of hitherto inaccessible proteins.

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Eluru, G., De Gieter, S., Schenck, S., Stroobants, A., Shrestha, B., Erbel, P., Brunner, J. D., Efremov, R. G.. 2025-01-14. MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony. https://doi.org/10.1101/2025.01.10.632437

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