Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins
Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins
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DOI：
10.1038/s41467-020-15363-0
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发表时间：
2020-03-27
影响因子：
16.6
通讯作者：
Kossiakoff, Anthony A.
中科院分区：
文献类型：
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作者：
Mukherjee, Somnath;Erramilli, Satchal K.;Kossiakoff, Anthony A.
我们提出了通用基准的概念，基于一组预制的半合成抗体（sAB）产生的定制噬菌体展示选择对融合蛋白BRIL，脱辅基细胞色素b562 a的工程变体。这些sAB可以结合到BRIL融合到不同GPCR、离子通道、受体和转运蛋白的环或末端中，而不破坏它们的结构。BRIL与亲和力成熟的sAB（BAG 2）复合的晶体结构描述了相互作用的足迹，所述sAB与所有测试的系统结合。BRIL-膜蛋白嵌合体的几个实例的负染色和cryoEM数据突出了sAB作为通用基准标记的有效性。结合sAB结合的人烟碱乙酰胆碱受体的cryoEM结构，这项工作表明，这些抗BRIL sAB可以大大增强颗粒性质，从而改善cryoEM结果，特别是对于挑战性膜蛋白。
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.