Biology subjects

Amyot, R.

Publications and source records attributed to Amyot, R..

8 recordsLinked to original sources

bioRxiv2026

BioAFMviewer AFM Analysis Arena A3 - an interactive software platform for automated analysis of HS-AFM imaging

High-speed atomic force microscopy (HS-AFM) experiments provide unique insights into nanoscale biological processes by directly visualizing the surface topography dynamics of biomolecules, such as proteins, over extended periods of time and generating large imaging data sets. However, the lack of computational methods for automated data analysis has thus far limited the full utilization of these data for the quantitative understanding of single-molecule dynamic. Here, we present the AFM Analysis Arena (A3) within BioAFMviewer as a software platform for automated, high-throughput quantitative analysis of AFM topographic imaging data within a user-friendly interactive graphical interface, implementing a convenient workflow from raw HS-AFM (AFM) imaging to statistical analysis. In several applications to HS-AFM imaging data, we demonstrate methods of automatized molecule detection and dynamic tracking to highlight the available capacities, aiming to transform HS-AFM measurements from observations towards an analytical tool in nanoscale biology.

biophysics↗

bioRxiv2026

Characterization of the trimeric TOM complex by HS-AFM single-molecule analysis

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial proteins. Approximately 99 % of mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and subsequently translocated into mitochondria through the TOM complex. The TOM complex exists in a dynamic equilibrium among multiple assembly states through spatial rearrangements of its subunits. The recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the TOM core dimer, whereas previous biochemical studies indicated the TOM complex functions as a trimer in intact mitochondria. However, the relationship between the core dimer and the functional trimer remains unclear. In the present study, we analyzed the dynamics of the TOM complex using high-speed atomic force microscopy (HS-AFM) to investigate the assembly states and conformation transitions of the TOM complexes. We demonstrated that purified yeast TOM complexes predominantly adopt a trimeric organization but dynamically dissociate into dimeric and monomeric states during HS-AFM observation. The trimeric particles observed by HS-AFM exhibited spherical molecular shapes consistent with a trimeric structural model proposed from previous crosslinking analyses. In contrast, the dissociated dimeric particles closely resembled the dimensions of the TOM core-dimer structures determined by cryo-EM. Furthermore, HS-AFM analyses provided insight into the spatial arrangement of the Tom20 receptor, consistent with previous models of the trimeric TOM complex. These observations enabled characterization of the trimeric TOM complex in vitro and provide a foundation for future structural and functional analyses of TOM complex assembly. Significance statementThe translocase of the mitochondrial outer membrane (TOM) complex is the universal entry gate for almost all mitochondrial proteins, yet its native organization has remained elusive. By combining high-speed atomic force microscopy with cryo-electron microscopy, we provide evidence that the yeast TOM complex predominantly adopts a trimeric assembly with three protein-conducting channels, rather than the dimeric architecture proposed by previous structural studies. Furthermore, single-molecule imaging directly visualized interactions with mitochondrial precursor proteins and receptor-specific antibodies, enabling the experimental identification of receptor subunits within the trimeric TOM complex. Together, these findings uncover previously unrecognized structural and dynamic features of the TOM complex and provide a framework for understanding the molecular mechanism of mitochondrial protein import.

biochemistry↗

bioRxiv2026

Coiled-coil domain kinking controls laminin-332 cleavage by elastase

Laminins are trimeric glycoproteins and essential components of basement membranes, where they provide structural support and mediate cell-matrix adhesion. Their -, {beta}-, and {gamma}-chains combine into a flexible trimeric coiled-coil domain forming the laminin long arm, which links the N-terminal short arms and the C-terminal globular domains. However, ultrastructural insight into the laminin coiled-coil domain remains limited, and the function of coiled-coil flexibility remains unclear. Using high-speed atomic force microscopy (HS-AFM) imaging, we previously investigated the ultrastructure of laminin-332 and observed dynamic kinking around a putative molecular hinge in the center of the coiled-coil domain. In this study, we integrate HS-AFM time-lapse imaging with AlphaFold structure prediction and normal-mode flexible fitting (NMFF) to generate dynamic models of the laminin-332 hinge during coiled-coil domain kinking, providing atomic-scale insight into the underlying molecular rearrangements. Furthermore, we use HS-AFM to visualize, in real time, the digestion of individual laminin-332 molecules by pancreatic elastase and show that coiled-coil kinking directs elastase cleavage to the hinge site, thereby ensuring reliable generation of the elastase 8 (E8) fragment containing the integrin binding site. In contrast, laminin molecules with extended coiled-coil conformations are cleaved at arbitrary sites, resulting in complete coiled-coil removal and degradation. Our results thus identify a novel conformational control mechanism directing proteolytic processing of laminin-332 through defined coiled-coil kinking. Besides expanding the understanding of dynamic laminin coiled-coil function, the gained ultrastructural insight into trimeric coiled-coil bending may aid the rational design of flexible coiled-coil modules for future protein engineering applications.

cell biology↗

bioRxiv2025

Flexible fitting to infer atomistic-precision models of large-amplitude conformational dynamics in biomolecules from high-speed atomic force microscopy imaging

High-speed atomic force microscopy (HS-AFM) experiments allow direct observation of biomolecular dynamics at the single-molecule level, acquiring a large amount of topographic imaging data that visualizes changes of the molecular surface during functional activity over an extended period of time. Since images have no atomistic resolution, a major challenge has been to develop post-experimental computational methods to infer atomistic information from measurements. The recently developed NMFF-AFM flexible fitting method provides a computationally efficient approach promising to infer atomistic-precision models of conformational dynamics from resolution-limited AFM imaging data. We report the software integration of this method into the well-established BioAFMviewer platform and demonstrate first applications to experimental HS-AFM imaging data. To facilitate applications, we developed a direct workflow from raw experimental AFM data to visualization and analysis of fitting results. The presented applications to experimental data of a single protein domain, a protein complex, and a megadalton size protein filament demonstrate versatility of NMFF-AFM modelling to reproduce large-amplitude conformational motions of biomolecular dynamics from HS-AFM imaging. As a first step towards automated large-scale analysis of AFM imaging data, we furthermore demonstrate reconstruction of an atomistic molecular movie of protein dynamics, involving large-amplitude conformational transitions, from a measured HS-AFM movie sequence. Implementation of flexible fitting within the standalone user-friendly interactive BioAFMviewer software opens the opportunity for broad range applications to facilitate the understanding of resolution-limited HS-AFM measurements.

biophysics↗

bioRxiv2023

Atom filtering algorithm and GPU-accelerated calculation of simulation atomic force microscopy images

Simulation atomic force microscopy computationally emulates experimental scanning of a biomolecular structure to produce topographic images that can be correlated with measured images. Its application to the enormous amount of available high-resolution structures, as well as to molecular dynamics modelling data, facilitates the quantitative interpretation of experimental observations by inferring atomistic information from resolution-limited measured topographies. The computation required to generate a simulated AFM image generally includes the calculation of contacts between the scanning tip and all atoms from the biomolecular structure. However, since only contacts with surface atoms are relevant, a filtering method shall highly improve the efficiency of simulation AFM computations. In this report we address this issue and present an elegant solution based on graphics processing unit (GPU) computations that significantly accelerates the computation of simulation AFM images. The method not only allows for visualization of biomolecular structures combined with ultra-fast synchronized calculation and graphical representation of corresponding simulated AFM images (live simulation AFM), but, as we demonstrate, can also reduce the computational effort during automatized fitting of atomistic structures into measured AFM topographies by orders of magnitude. Hence, the developed method will play an important role in post-experimental computational analysis involving simulation AFM. Implementation is realized in our BioAFMviewer software package for simulation AFM of biomolecular structures and dynamics.

biophysics↗

bioRxiv2023

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, and the ClpB molecular chaperone. The model also allows predictions of imaging stability under tip-scanning. We implemented the developed method within the freely available BioAFMviewer software package. Therefore, predictions based on available structural data can be made even prior to an actual experiment.

biophysics↗

bioRxiv2021

Atomic reconstruction of biomolecular structures from AFM images and quantitative validation of experimental data using simulated AFM scanning

We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.

biophysics↗

bioRxiv2019

Biomolecular AFM viewer

We developed a stand-alone software which allows to transform biomolecular structures and movies of their conformational dynamics into a representation which corresponds to the outcome of biomolecular atomic force microscopy (AFM) experiments, such as high-speed AFM. The software implements a high degree of usability. An integrated highly versatile molecular viewer allows the visualization of structures and their corresponding simulated AFM representations in arbitrary orientations. The obtained results can be conveniently exported as still images and movies. We provide a demonstration of our biomolecular AFM viewer by applying it to several proteins from the Protein Data Bank, and to a molecular movie of conformational transitions between two protein structures obtained from a modelling server.

biophysics↗