德国欧洲分子生物学实验室Julia Mahamid等研究人员，合作开发出用于cryo-EM亚细胞蛋白质定位的遗传编码多聚标签。该研究于2023年11月6日在线发表于国际一流学术期刊《自然—方法学》。

研究人员提出了一种配体诱导型细胞内蛋白质标记策略，该策略基于25纳米大小的荧光遗传编码多聚体颗粒（GEM）。这些颗粒具有可识别的结构特征，可通过卷积神经网络低温电子断层成像（cryo-ET）数据中对其进行自动检测。通过添加小分子配体触发GEM与绿色荧光蛋白标记的相关大分子的耦合，从而实现时间可控的标记，最大限度地减少对原生蛋白功能的干扰。

研究人员利用低温相关荧光和cryo-ET成像技术展示了GEM在人体细胞不同细胞器中，对内源性和过表达蛋白质进行亚细胞级定位的适用性。研究人员介绍了量化标记特异性和效率的方法，以及针对稀有和丰富蛋白质靶点进行系统优化的方法，其重点是评估标记对蛋白质功能的潜在影响。

据悉，cryo-ET可对原生细胞环境中的大分子组装体进行无标记高分辨率成像。然而，在层析体积中对感兴趣的大分子进行定位是一项挑战。

附：英文原文

Title: Genetically encoded multimeric tags for subcellular protein localization in cryo-EM

Author: Fung, Herman K. H., Hayashi, Yuki, Salo, Veijo T., Babenko, Anastasiia, Zagoriy, Ievgeniia, Brunner, Andreas, Ellenberg, Jan, Mller, Christoph W., Cuylen-Haering, Sara, Mahamid, Julia

Issue&Volume: 2023-11-06

Abstract: Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function. Genetically encoded multimeric particles (GEMs) are 25-nm tags with recognizable structural signatures, which can be used to label specific proteins in mammalian cells to facilitate their subcellular localization in cryo-ET.

DOI: 10.1038/s41592-023-02053-0

Source: https://www.nature.com/articles/s41592-023-02053-0