Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy.

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter‐ and intra‐molecular interactions. Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes. Furthermore, the principle of protein assembly gives guidelines for new biomimetic materials with potential applications in medicine, energy, and nanotechnology. Atomic force microscopy (AFM) is a powerful tool for investigating protein assembly and interactions across spatial scales (single molecules to cells) and temporal scales (milliseconds to days). It has significantly contributed to understanding nanoscale architectures, inter‐ and intra‐molecular interactions, and regulatory elements that determine protein structures, assemblies, and functions. This review describes recent advancements in elucidating protein assemblies with in situ AFM. We discuss the structures, diffusions, interactions, and assembly dynamics of proteins captured by conventional and high‐speed AFM in near‐native environments and recent AFM developments in the multimodal high‐resolution imaging, bimodal imaging, live cell imaging, and machine‐learning‐enhanced data analysis. These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research. [ABSTRACT FROM AUTHOR]