Affinity grids have great potential to facilitate rapid preparation of even quite impure samples in single-particle cryo-electron microscopy (EM). Yet despite the promising advances of affinity grids over the past decades, no single strategy has demonstrated general utility. Here we chemically functionalize cryo-EM grids coated with mostly one or two layers of graphene oxide to facilitate affinity capture. The protein of interest is tagged using a system that rapidly forms a highly specific covalent bond to its cognate catcher linked to the grid via a polyethylene glycol (PEG) spacer. Importantly, the spacer keeps particles away from both the air–water interface and the graphene oxide surface, protecting them from potential denaturation and rendering them sufficiently flexible to avoid preferential sample orientation concerns. Furthermore, the PEG spacer successfully reduces nonspecific binding, enabling high-resolution reconstructions from a much cruder lysate sample.

亲和网格具有巨大的潜力，可促进单粒子低温电子显微镜（EM）中甚至非常不纯的样品的快速制备。尽管在过去的几十年中，亲和网格取得了令人鼓舞的进步，但没有哪一种策略能显示出通用性。在这里，我们对大部分涂有一层或两层氧化石墨烯的低温EM栅格进行化学功能化，以促进亲和力捕获。使用快速标记一个高特异性共价键的系统来标记目的蛋白质，该系统通过聚乙二醇（PEG）间隔子与网格连接。重要的是，隔离片使微粒远离空气-水界面和氧化石墨烯表面，从而保护它们免受潜在的变性并使其具有足够的柔韧性，从而避免了优先考虑的样品取向问题。此外，