National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 100101, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Nat Commun. 2021 Dec 14;12(1):7257. doi: 10.1038/s41467-021-27596-8.
Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.
冷冻电子显微镜（cryo-EM）已成为解析溶液中生物大分子高分辨率结构的有力工具。然而，在冷冻玻璃化过程中，空气-水界面诱导的生物大分子优先取向、解离或变性仍然是许多样本的限制因素。为了解决这个瓶颈问题，我们开发了一种使用亲脂性抗菌肽 HFBI 二维晶体的 cryo-EM 支撑膜。HFBI 膜的亲水侧通过静电相互作用吸附蛋白质颗粒，并将其与空气-水界面隔离，从而形成足够薄的冰层，以进行高质量的数据收集。通过调节缓冲液 pH 值可以调节颗粒的取向分布。使用这种支撑膜，我们确定了过氧化氢酶（2.29 Å）和流感血凝素三聚体（2.56 Å）的 cryo-EM 结构，它们在使用传统 cryo-vitrification 方案时表现出强烈的优先取向。我们进一步表明，HFBI 膜适用于获得小蛋白的高分辨率结构，包括醛缩酶（150 kDa，3.28 Å）和血红蛋白（64 kDa，3.6 Å）。我们的工作表明，HFBI 膜可能在提高 cryo-EM 的成功率和效率方面具有广泛的未来应用。
