The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297–391), termed ‘dGAE’. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are structurally closely related to the paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.
Bibliographical noteFunding Information:
We thank Michel Goedert and Sjors Scheres for insightful discussions, and the members of the Xue group, the Kent Fungal Group and the Louise Serpell group for helpful comments throughout the preparation of this manuscript. This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC), UK grant BB/S003312/1 (WFX and LCS), as well as Engineering and Physical Sciences Research Council (EPSRC), UK DTP grant EP/R513246/1 (LL). Research in the LCS lab, and YA-H is supported by funding from WisTa Laboratories Ltd (PAR1596).
- atomic force microscopy
- cryo-electron microscopy
- structural biology