Deciphering the three-dimensional structures of macromolecules is of paramount importance for gaining insights into their functions and roles in human health and disease. Single particle cryoEM has emerged as a powerful technique that enables direct visualization of macromolecules and their complexes, and through subsequent averaging, achieve near atomic-level resolution. A major breakthrough was recently achieved with the determination of the apoferritin structure at true atomic resolution. In this review, we discuss the latest technological innovations across the entire single-particle workflow, which have been instrumental in driving and expanding the resolution revolution and in transforming cryoEM as a mainstream technique in structural biology. We illustrate these advancements using apoferritin as an example that has served as an excellent benchmark sample for assessing emerging technologies. We further explore whether the existing technology can routinely generate atomic structures of dynamic macromolecules that more accurately represent real-world samples, the limitations in the workflow and the current approaches employed to overcome them.

中文翻译：

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使用脱铁铁蛋白探索单粒子 CryoEM 的进展：从斑点到真正的原子分辨率

破译大分子的三维结构对于深入了解它们在人类健康和疾病中的功能和作用至关重要。单粒子冷冻电镜已经成为一种强大的技术，可以直接可视化大分子及其复合物，并通过随后的平均，实现接近原子级的分辨率。最近以真正的原子分辨率测定脱铁铁蛋白结构，取得了重大突破。在这篇综述中，我们讨论了整个单粒子工作流程中的最新技术创新，这些创新在推动和扩大分辨率革命以及将冷冻电镜转变为结构生物学的主流技术方面发挥了重要作用。我们以去铁铁蛋白为例来说明这些进步，该蛋白已成为评估新兴技术的优秀基准样本。我们进一步探讨现有技术是否可以常规地生成动态大分子的原子结构，更准确地代表现实世界的样品、工作流程的局限性以及当前用于克服这些局限性的方法。