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bioRxiv · 10.1101/2021.11.09.467987

A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

Fan, H.·Wang, B.·Zhang, Y.·Zhu, Y.·Song, B.·Xu, H.·Zhai, Y.·Qiao, M.·Sun, F.
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Abstract

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28-[A]) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34-[A]) and hemoglobin (64 kDa, 3.6-[A]). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

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Fan, H., Wang, B., Zhang, Y., Zhu, Y., Song, B., Xu, H., Zhai, Y., Qiao, M., Sun, F.. 2021-11-11. A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein. https://doi.org/10.1101/2021.11.09.467987

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