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

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.