Yujia Qiu, Shinnosuke Narimatsu, Elma Sakinatus Sajidah, Keesiang Lim, Richard W Wong
1Division of Nano Life Science, Graduate School of Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
2WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan
3Cell-Bionomics Research Unit, Innovative Integrated Bio-Research Core, Institute for Frontier Science Initiative, Kanazawa University, Kanazawa 920-1192, Japan
Nuclear pore complexes (NPCs) on the nuclear membrane surface govern the flow of small and macromolecules between the cell nucleus and cytoplasm through their complicated core channel, which resembles a spiderweb. Few nanoscale technologies exist to reliably evaluate nuclear pore dynamics on nuclear membranes. Traditional optical imaging cannot resolve a variety of organelles and proteins in cells owing to diffraction. By allowing nanoscale subcellular inspection, super-resolution methods have overcome this restriction. However, fixed samples are generally needed for these approaches. This also raises the question of how well a static picture portrays intracellular dynamics. The unique technology of high-speed atomic force microscopy (HS-AFM) allows dynamic structural biology researchers to investigate molecules in motion near to their natural states. We use HS-AFM, which provides simultaneous structural and temporal resolution, to study protein kinetic mode switching. We created Y-shaped protein reconstitution for subcomplex and molecular analysis. With detection of local maxima, height extraction, merging of height modules from the localization atomic force microscopy (LAFM) and our novel JAVA coding, we could link structural states to a functional chronology and solve structures from a single molecule. Our novel modification approaches enable the development of more sophisticated time-resolved dynamic single-molecule structural methods for discovering structures using AFM.