Logo Logo
Switch Language to German

Konrad, Sebastian F.; Vanderlinden, Willem and Lipfert, Jan (2021): A High-throughput Pipeline to Determine DNA and Nucleosome Conformations by AFM Imaging. In: Bio-Protocol, Vol. 11, No. 19, e4180

Full text not available from 'Open Access LMU'.


Atomic force microscopy (AFM) is a powerful tool to image macromolecular complexes with nanometer resolution and exquisite single-molecule sensitivity. While AFM imaging is well-established to investigate DNA and nucleoprotein complexes, AFM studies are often limited by small datasets and manual image analysis that is slow and prone to user bias. Recently, we have shown that a combination of large scale AFM imaging and automated image analysis of nucleosomes can overcome these previous limitations of AFM nucleoprotein studies. Using our high-throughput imaging and analysis pipeline, we have resolved nucleosome wrapping intermediates with five base pair resolution and revealed how distinct nucleosome variants and environmental conditions affect the unwrapping pathways of nucleosomal DNA. Here, we provide a detailed protocol of our workflow to analyze DNA and nucleosome conformations focusing on practical aspects and experimental parameters. We expect our protocol to drastically enhance AFM analyses of DNA and nucleosomes and to be readily adaptable to a wide variety of other protein and protein-nucleic acid complexes.

Actions (login required)

View Item View Item