The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about 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 prototypical 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 apo-ferritin, our strategy led to a 1.2 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. Combination of the technological advances described here 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.

中文翻译：

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原子分辨率的单粒子冷冻EM

蛋白质分子中原子的三维位置定义了它们的结构，并对它们在复杂的生物过程中所扮演的角色提供了机械的见解。确定越精确的原子坐标，就可以得到越多的化学信息，并且就可以推断出更多关于蛋白质功能的知识。随着电子检测和图像处理技术的突破，近年来，电子冷冻显微镜（cryo-EM）单颗粒分析已产生了蛋白质结构，其详细程度不断提高。然而，迄今为止，获得具有足够分辨率以可视化蛋白质中单个原子的冷冻-EM重建技术还很困难。在这里，我们证明了使用新的电子源，能量过滤器和照相机，我们得到了1。原型人膜蛋白β3GABAA受体同戊四聚体的7Å分辨率cryo-EM重建。此类图谱可让您详细了解小分子配位，溶剂分子的可视化以及多种氨基酸的替代构象，以及明确构建有序的酸性侧链和聚糖。将其应用于小鼠脱铁铁蛋白后，我们的策略导致了1.2Å分辨率的重建，这是首次使用单粒子冰冻EM提供了蛋白质分子的真正原子分辨率视图。而且，许多氢原子的散射电位可以在差异图中显示，从而可以直接分析氢键网络。