Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes ‘magnification’ of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.</jats:p>

Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

“…3D modelling of fibril structures. Straightened fibril traces were corrected for the tip-convolution effect using an algorithm based on geometric modelling of the tipfibril contact points 72 . Briefly, the variation in the tip radius from their nominal value was first determined.…”

Heterotypic Aβ interactions facilitate amyloid assembly and modify amyloid structure

“…Nanoscope analysis software (Version 1.5, Bruker) were used to process the image data by flattening the height topology data to remove tilt and scanner bow. Fibrils were traced (Aubrey et al , 2020; Xue et al , 2009), digitally straightened(Egelman, 1986), and surface envelope reconstructed as previously described(Aubrey et al , 2020; Lutter et al , 2020) using an in-house application. The height profile for each fibril was extracted from the centre contour line of the straightened fibrils from which the average height of each fibril was calculated.…”

Rigid-body fitting to atomic force microscopy images for inferring probe shape and biomolecular structure

“…Although TMV is a stable and easy-to-use calibrator, still it is not always easy to observe it at the same condition as other molecules that generally requires different conditions to be stabilized on the AFM stage. Also, a recent work [26] that reconstructs the three-dimensional structures of amyloid fibril developing a method to refine the topography carefully estimates the probe tip radius. However, they use the ideal corkscrew symmetry in the specimen and it is not always applicable to other biomolecules.…”