Abstract：
Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.
摘要：
单粒子低温电子显微镜（cryo-EM）已成为一种基本的结构确定技术，最近的硬件发展使达到原子分辨率成为可能。单个原子，包括氢原子，可以解决。在这项研究中,我们使用核黄素生物合成倒数第二步中涉及的酶作为测试样本，对最近安装的显微镜进行基准测试，并确定其他蛋白质复合物是否可以达到1.5或更高的分辨率，到目前为止，只有铁载体铁蛋白才能实现。使用最先进的显微镜和检测器硬件以及最新的软件技术来克服显微镜和样品的限制，从48小时的显微镜观察中获得了Aquifexaeolicuslumazine合酶（AaLS）的1.42的图。除了参与AaLS功能的水分子和配体，我们可以观察到50%的氢原子的正密度。通过Ewald球体校正可以实现分辨率的小幅提高，预计该直径的分子将分辨率限制在〜1.5。我们的研究证实，其他蛋白质复合物可以解决近原子分辨率。未来在标本制备和蛋白质复合物稳定方面的改进可能会使更灵活的大分子达到这种分辨率水平，并应成为该领域研究的重点。
