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Structure determination from lipidic cubic phase embedded microcrystals by MicroED

By Lan Zhu, Guanhong Bu, Liang Jing, Dan Shi, Tamir Gonen, Wei Liu, Brent L. Nannenga

Posted 04 Aug 2019
bioRxiv DOI: 10.1101/724575 (published DOI: 10.1016/j.str.2020.07.006)

The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with well-diffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP.

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