Single-particle cryo-EM at atomic resolution.
Single-particle cryo-EM at atomic resolution.
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DOI：
10.1038/s41586-020-2829-0
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发表时间：
2020-11
期刊：
影响因子：
64.8
通讯作者：
Scheres SHW
中科院分区：
文献类型：
--
作者：
Nakane T;Kotecha A;Sente A;McMullan G;Masiulis S;Brown PMGE;Grigoras IT;Malinauskaite L;Malinauskas T;Miehling J;Uchański T;Yu L;Karia D;Pechnikova EV;de Jong E;Keizer J;Bischoff M;McCormack J;Tiemeijer P;Hardwick SW;Chirgadze DY;Murshudov G;Aricescu AR;Scheres SHW
蛋白质分子中原子的三维位置决定了它们的结构和它们在生物过程中的作用。确定的原子坐标越精确，就可以得到越多的化学信息，并且可以推断出对蛋白质功能的更多的机械见解。近年来，随着电子检测和图像处理技术的突破，电子冷冻显微镜（cryo-EM）单粒子分析已经产生了越来越多的细节水平的蛋白质结构。然而，迄今为止，获得具有足够分辨率的cryo-EM重建以可视化蛋白质中的单个原子是难以捉摸的。在这里，我们表明，使用一种新的电子源，能量过滤器和相机，我们获得了1.7 μ m分辨率的冷冻-EM重建的人膜蛋白，β3 GABAA受体homopentamer。这样的地图可以详细了解小分子的协调，可视化的溶剂分子和多种氨基酸的替代构象，以及明确的建设有序的酸性侧链和聚糖。应用于小鼠脱铁铁蛋白，我们的策略导致了1.22 μ m分辨率的重建，这是第一次，提供了一个真正的原子分辨率的蛋白质分子使用单粒子冷冻EM。此外，许多氢原子的散射势可以在差分图中可视化，从而可以直接分析氢键网络。这里描述的技术进步，结合进一步的方法，以加速数据采集和提高样品质量，提供了一条路线，在高通量筛选小分子调节剂和基于结构的药物发现的常规应用cryo-EM。
The three-dimensional positions of atoms in protein molecules define their structure and the roles they perform in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer. Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. The technological advances described here, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.