Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC 3800, Australia.
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, VIC 3800, Australia.
J Struct Biol. 2019 Sep 1;207(3):327-331. doi: 10.1016/j.jsb.2019.07.004. Epub 2019 Jul 16.
Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.
低温电子显微镜（cryo-EM）允许对由于其大小和异质性而抵制其他结构生物学方法的大分子组装进行结构测定。这些具有挑战性的多蛋白靶标通常在 cryo-EM 网格制备时容易发生解离和/或变性，并且通常需要在冷冻之前交联。已经开发了几种温和的柱上或管内交联方法。由于凝胶过滤技术的分离性能差，柱上交联的应用并不广泛。管内交联经常导致样品聚集和/或沉淀。通过 GraFix 方法进行基于梯度的交联更稳健，但非常耗时，并且需要专门的昂贵设备。此外，甘油的去除通常会导致大量样品损失，并可能导致对样品质量有害的失稳。在这里，我们介绍一种替代方法：AgarFix（琼脂糖固定）。将样品嵌入琼脂糖基质中，使分子保持分离，从而防止交联时形成聚集体。通过将交联剂扩散到琼脂糖滴中来实现温和的交联。通过扩散或电洗脱回收样品，可直接用于 cryo-EM 标本制备。AgarFix 需要最少的设备和基本的实验室经验，使其广泛适用于 cryo-EM 社区。
