Structural Identification of Individual Helical Amyloid Filaments by Integration of Cryo-Electron Microscopy-Derived Maps in Comparative Morphometric Atomic Force Microscopy Image Analysis

Abstract

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.

Keywords: amyloid; atomic force microscopy; cryo-electron microscopy; polymorphism; structural biology.

Figure 1

Analysis of individual dGAE filaments from a topographical AFM height image. (a) A typical AFM image of dGAE fibrils with an inset showing a 2× magnified view. Arrows point to the fibrils shown in (b) and (c). Scale bar represents 500 nm. (b) Digitally straightened images showing a typical dGAE fibril and typical fibril bundles. Scale bar represents 200 nm. (c) Digitally straightened AFM image of a canonical dGAE tau fibril together with a 3D reconstructed surface envelope model. Scale bar represents 50 nm. (d) The central line height profile of the fibril image shown in (c).

Figure 2

Simulation of topographic AFM height images from cryo-EM density maps. Cross-sectional views of axis-aligned cryo-EM density map iso-surfaces for (a) paired helical filaments (EMD-0259) and (c) ‘heparin-snake’ (EMD-4563) tau fibrils are shown with the position of the helical axis (+). Simulated topographic images of 500 nm fibril segments and the top view of extended 250 nm segments of iso-surfaces aligned to the simulated images are shown for PHF in (b) and ‘heparin-snake’ in (d). All scale bars represent 10 nm.

Figure 3

Comparison of the typical experimental AFM height image of dGAE tau fibril with topographical images of tau amyloid fibril polymorphs. The tau fibril polymorphs were simulated with identical tip (radius here estimated to be 11.7 nm) and sampling parameters using extended and axis-aligned cryo-EM map iso-surfaces. The ‘*’ indicate image data shown in Figure 1(c). Length of the scale bar is 50 nm. The simulated images have been aligned to the same estimated helical axis height position as the experimental data image and, therefore, have comparable height colour coding. The EMDB accession codes of the cryo-EM density maps from which the topographs were simulated from are listed with the abbreviated names of the individual filaments. The sample origin is noted above the filaments’ images. The following abbreviations were used for disease diagnoses and filament labels: CBD – corticobasal degeneration, CTE – chronic traumatic encephalopathy, PiD – Pick’s disease, AD – Alzheimer’s disease, NPF – narrow Pick filament, WPF – wide Pick filament, PHF – paired helical filament, SF – straight filament.

Figure 4

Comparison of the cross-section of the surface envelope model derived from the experimental AFM dGAE filament image and cryo-EM density map cross-sections. The cross-sections for the CTE type II filaments (a) and PHF from AD patient tissue (b), as well as in vitro assembled heparin-induced ‘twister’ filaments (c) are shown. The average cross-section of the AFM image derived 3D model is shown as thick blue lines, and the structural variation observed along the fibril in the AFM image is indicated by the thin blue lines denoting the 2.5 and 97.5 percentile bounds. The cryo-EM density cross-sections are shown in grey.

Figure 5

The image similarity score and the combined score of similarity between the dGAE fibril AFM image data and cryo-EM derived density maps of tau fibril polymorphs. Lower score denotes higher degree of similarity. The overall comparative rankings (from highest to lowest) based on the combined score of similarity (Supplementary Table S1) are labelled for each entry.