Macromolecular protein assemblies are of fundamental importance for many processes inside the cell, as they perform complex functions and constitute central hubs where reactions occur. Generally, these assemblies undergo large conformational changes and cycle through different states that ultimately are connected to specific functions further regulated by additional small ligands or proteins. Unveiling the 3D structural details of these assemblies at atomic resolution, identifying the flexible parts of the complexes, and monitoring with high temporal resolution the dynamic interplay between different protein regions under physiological conditions is key to fully understanding their properties and to fostering biomedical applications.

In the last decade, we have seen remarkable advances in cryo-electron microscopy (EM) techniques, which deeply transformed our vision of structural biology, especially in the field of macromolecular assemblies. With cryo-EM, detailed 3D models of large macromolecular complexes in different conformational states became readily available at atomic resolution. Concomitantly, nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy (EPR) have benefited from methodological innovations which also improved the quality of the information that can be achieved. Such enhanced sensitivity widened their applicability to macromolecular complexes in environments close to physiological conditions and opened a path towards in-cell applications.

In this review we will focus on the advantages and challenges of EPR techniques with an integrative approach towards a complete understanding of macromolecular structures and functions.

大分子蛋白质组装体对于细胞内的许多过程至关重要，因为它们执行复杂的功能并构成反应发生的中心枢纽。一般来说，这些组装体会经历较大的构象变化，并在不同的状态之间循环，最终与额外的小配体或蛋白质进一步调节的特定功能相关。以原子分辨率揭示这些组件的 3D 结构细节，识别复合物的柔性部分，并以高时间分辨率监测生理条件下不同蛋白质区域之间的动态相互作用，是充分了解其特性和促进生物医学应用的关键。

在过去的十年中，我们看到了冷冻电子显微镜（EM）技术的显着进步，它深刻地改变了我们对结构生物学的看法，特别是在大分子组装领域。借助冷冻电镜，可以轻松获得不同构象状态的大分子复合物的详细 3D 模型（原子分辨率）。与此同时，核磁共振（NMR）和电子顺磁共振波谱（EPR）也受益于方法创新，这也提高了可获得的信息的质量。这种增强的灵敏度扩大了它们在接近生理条件的环境中对大分子复合物的适用性，并为细胞内应用开辟了道路。

在这篇综述中，我们将重点关注 EPR 技术的优势和挑战，并采用综合方法来全面了解大分子结构和功能。