人源MDN1蛋白质的电镜结构研究

doi:10.3969/j.issn.1674-8115.2021.05.001

摘要/Abstract

摘要：

目的·利用负染电镜技术分析人源midasin AAA-ATPase 1（MDN1，又称Rea1）蛋白质结构。方法·利用CRISPR/Cas9基因编辑方法在Expi293F细胞内源MDN1蛋白质的氨基端敲入3×FLAG纯化标签，采用ANTI-FLAG^? M2 Agarose Affinity Gel亲和层析和甘油密度梯度离心的方法分离纯化目的蛋白质；通过负染色电镜技术和单颗粒（single-particle）重构技术探究人源MDN1蛋白质结构。结果·利用亲和层析及密度梯度离心方法分离纯化获得高纯度、均一性较好的人源MDN1蛋白质样品；采用甲酸铀负染色后利用120 kV电镜初步解析了目的蛋白质MDN1的空间结构。结论·利用单颗粒重构技术搭建了人源MDN1蛋白质的低分辨率的负染模型。

关键词: MDN1蛋白质, pre-60S核糖体, 核糖体成熟, 负染电镜

Abstract:

Objective·To study the structure of the human midasin AAA-ATPase 1 (MDN1, Rea1) protein by negative-staining electron microscopy.

Methods·Using the CRISPR/Cas9 genome editing method, a 3×FLAG affinity tag was inserted into the N-terminus of MDN1 in Expi293F cells. Tagged proteins were isolated via affinity purification with ANTI-FLAG^? M2 Agarose Affinity Gel, followed by glycerol density gradient centrifugation. The purified protein sample was then subjected to negative-staining electron microscopy and single particle image analysis.

Results·The FLAG-tagged endogenous MDN1 proteins with high purity and good homogeneity were obtained using affinity chromatography and density gradient centrifugation. Preliminary study on the structure of human MDN1 was achieved by 120 kV electron microscope after negative staining with uranium formate.

Conclusion·A low resolution model of human MDN1 protein was achieved by single particle reconstruction analysis.

Key words: MDN1 protein, pre-60S ribosome, ribosome maturation, negative-staining electron microscopy

中图分类号:

Q518.2

许云涛, 李明月, 雷鸣. 人源MDN1蛋白质的电镜结构研究[J]. 上海交通大学学报(医学版), 2021, 41(5): 559-564.

Yun-tao XU, Ming-yue LI, Ming LEI. Electron microscopic study of the human MDN1 protein[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(5): 559-564.

图/表 3

图1 人源内源FLAG-MDN1蛋白质纯化Note: A. Schematic representation of MDN1 domain organization. FLAG tag was inserted into the N-terminus of MDN1. B/C. Verification of the knock-in of FLAG tag by PCR (B) and Western blotting （C）. NC—negative control; F—FLAG tag knock-in cell lines. The arrow indicates expected PCR products or protein bands. D. IF detection of endogenous FLAG-tagged MDN1 protein. E/F. Western blotting analysis and silver staining of purified FLAG-MDN1. G. Silver staining of protein fractions after density gradient centrifugation. 1?20—protein fractions.

Fig 1 Purification of the human endogenous FLAG-tagged MDN1 protein

图2 负染电镜分析MDN1蛋白质Note: A. A representative negative-staining electron micrograph image of MDN1. B. Selected reference-free 2D class averages. C. Reconstruction of MDN1 protein shown in six orthogonal views as indicated. D. Angular distribution of the particles used for the final reconstruction.

Fig 2 Negative-staining electron microscopy analysis of MDN1

图3 MDN1蛋白质三维模型和AAA结构域的定位Note: A. Gold standard Fourier shell correlation (FSC) curve of the final reconstructed electron micrograph map of MDN1. B. Surface view of the electron micrograph map of MDN1 and its volume size. C. Predicted atomic model of MDN1 AAA domains (amino acids—307-2313). D. Docking of the predicted atomic model of AAA domains into corresponding mass of MDN1 shown in two orthogonal views.

Fig 3 Three-dimensional reconstruction of MDN1 and localization of AAA domains

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