Kelly Deborah F, Dukovski Danijela, Walz Thomas
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4703-8. doi: 10.1073/pnas.0800867105. Epub 2008 Mar 17.
Visualizing macromolecular complexes by single-particle electron microscopy (EM) entails stringent biochemical purification, specimen preparation, low-dose imaging, and 3D image reconstruction. Here, we introduce the "monolayer purification" method, which employs nickel-nitrilotriacetic acid (Ni-NTA) functionalized lipids for simultaneously purifying His-tagged complexes directly from cell lysates while producing specimens suitable for single-particle EM. The method was established by using monolayers containing Ni-NTA lipid to specifically adsorb His-tagged transferrin-transferrin receptor (Tf-TfR) complexes from insect and mammalian cell extracts. The specificity and sensitivity of the method could be improved by adding imidazole to the extracts. The monolayer-purified Tf-TfR samples could be vitrified and used to calculate a 3D reconstruction of the complex. Monolayer purification was then used to rapidly isolate ribosomal complexes from bacteria by overexpressing a single His-tagged ribosomal subunit. The resulting monolayer samples allowed calculation of a cryo-EM 3D reconstruction of the Escherichia coli 50S ribosomal subunit.
通过单颗粒电子显微镜（EM）对大分子复合物进行可视化分析需要严格的生化纯化、样品制备、低剂量成像以及三维图像重建。在此，我们介绍“单层纯化”方法，该方法采用镍-次氮基三乙酸（Ni-NTA）功能化脂质，用于直接从细胞裂解物中同时纯化带有组氨酸标签的复合物，同时制备适用于单颗粒EM的样品。该方法是通过使用含有Ni-NTA脂质的单层来特异性吸附来自昆虫和哺乳动物细胞提取物中带有组氨酸标签的转铁蛋白-转铁蛋白受体（Tf-TfR）复合物而建立的。通过向提取物中添加咪唑可以提高该方法的特异性和灵敏度。单层纯化的Tf-TfR样品可以进行玻璃化处理，并用于计算该复合物的三维重建。然后通过过表达单个带有组氨酸标签的核糖体亚基，利用单层纯化从细菌中快速分离核糖体复合物。所得的单层样品可用于计算大肠杆菌50S核糖体亚基的冷冻电镜三维重建。
