Rames Matthew, Yu Yadong, Ren Gang
Lawrence Berkeley National Laboratory, The Molecular Foundry.
Lawrence Berkeley National Laboratory, The Molecular Foundry;
J Vis Exp. 2014 Aug 15(90):e51087. doi: 10.3791/51087.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
对于分子量在40 - 200 kDa之间的蛋白质，其结构测定颇具挑战性。鉴于超过一半的天然蛋白质分子量在40 - 200 kDa之间，因此需要一种强大的、具有纳米分辨率能力的高通量方法。负染色（NS）电子显微镜（EM）是一种简便、快速且定性的方法，在研究实验室中经常用于研究蛋白质结构和蛋白质 - 蛋白质相互作用。不幸的是，传统的NS方案常常会在蛋白质上产生结构假象，尤其是对于通常会形成缗钱状假象的脂蛋白。通过将低温电子显微镜（cryo - EM）得到的脂蛋白图像作为标准，最近筛选了NS样品制备条件中的关键参数，并报告了优化的NS方案（OpNS），这是一种改进的传统NS方案。OpNS可以极大地限制诸如缗钱状等假象的产生，此外还能提供高对比度以及小的不对称蛋白质的相当高分辨率（接近1 nm）图像。这些高分辨率和高对比度的图像甚至有利于通过电子断层扫描方法对单个蛋白质（单个对象，无平均值）进行三维重建，例如160 kDa的抗体。此外，OpNS可以作为一种高通量工具来检测数百个小蛋白质样品。例如，先前发表的53 kDa胆固醇酯转移蛋白（CETP）的机制涉及对数百个样品的筛选和成像。鉴于cryo - EM很少能成功成像小于200 kDa的蛋白质，且尚未发表任何涉及筛选一百多个样品条件的研究，将OpNS称为研究小蛋白质的高通量方法是合理的。希望这里介绍的OpNS方案能够成为推动电子显微镜边界、加速对小蛋白质结构、动力学和机制的电子显微镜研究的有用工具。
