We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

中文翻译：

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抗BRIL的合成抗体作为通用的基准标记，可用于确定膜蛋白的单粒子cryoEM结构。

我们提出了基于一组预先设定的半合成抗体（sAB）的通用基准的概念，这些抗体是通过针对融合蛋白BRIL（脱细胞色素b562a的工程变体）的定制噬菌体展示选择产生的。这些sABs可以结合到融合在不同GPCR，离子通道，受体和转运蛋白的环或末端中的BRIL，而不会破坏其结构。BRIL的晶体结构与亲和力成熟的sAB（BAG2）结合并结合到所有测试的系统上，描绘了相互作用的足迹。几个BRIL膜蛋白嵌合体实例的负染色和cryoEM数据突出了sAB作为通用基准标记的有效性。结合结合了sAB的人烟碱型乙酰胆碱受体的cryoEM结构，