比利时 VIB-VUB结构生物学中心Jan Steyaert和英国剑桥大学MRC分子生物学实验室A. Radu Aricescu研究团队合作的最新研究表明，由纳米抗体衍生的Megabodies工具包有助于通过单粒子冷冻电镜确定蛋白质结构。2021年1月6日，国际学术期刊《自然-方法学》发表了这一成果。

在本研究中，研究人员发现工程化的纳米抗体还可以帮助克服影响单粒子冷冻电镜重构分辨率的两个主要的障碍：粒径和在水-空气界面处的优先取向。研究人员通过将纳米抗体嫁接到选定的蛋白质支架上以增加其分子量，同时保留完整的抗原结合特异性和亲和力，从而开发并表征为megabody构建体。研究表明，megabody设计原则适用于不同的支架蛋白和兼容的几何结构识别域，并适合从酵母展示库中进行有效选择。

此外，研究还证明了megabody可用于膜蛋白的三维重构，这些膜蛋白具有强烈的优先取向或因分子量太小而无法进行精确的颗粒排列。

据悉，纳米抗体是结构生物学流行且通用的工具。它们具有紧凑的单个免疫球蛋白结构域，并以高亲和力结合靶蛋白，同时减少构象异质性并稳定多蛋白复合物。

附：英文原文

Title: Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Author: Tomasz Uchaski, Simonas Masiulis, Baptiste Fischer, Valentina Kalichuk, Uriel Lpez-Snchez, Eleftherios Zarkadas, Miriam Weckener, Andrija Sente, Philip Ward, Alexandre Wohlknig, Thomas Zgg, Han Remaut, James H. Naismith, Hugues Nury, Wim Vranken, A. Radu Aricescu, Els Pardon, Jan Steyaert

Issue&Volume: 2021-01-06

Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water–air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

DOI: 10.1038/s41592-020-01001-6

Source: https://www.nature.com/articles/s41592-020-01001-6