High-speed atomic force microscopy for dynamic single molecule structural biology.

Abstract

High-speed atomic force microscopy (HS-AFM) is a powerful technique that provides dynamic movies of biomolecules at work. We successfully used HS-AFM to take movies and determine dynamic parameters of membrane trafficking systems, transporters and channels. To break current temporal limitations to characterize molecular dynamics using HS-AFM, we developed HS-AFM line scanning (HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS), methods whereby we scan the HS-AFM tip along a single scan line or keep it at a fixed position, respectively, and detect the motions of the molecules under the tip with sub-nanometer spatial and millisecond to microseconds temporal resolution to characterize single molecule kinetics. To break resolution limitations, we developed Localization AFM (LAFM) by applying localization image reconstruction algorithms to peak positions in high-speed AFM and conventional AFM data and increased the resolution beyond the limits set by the tip radius to reach quasi-atomic resolution on soft protein surfaces in native and dynamic conditions. Here, I will review these recent achievements and present novel data and methods to integrate and strengthen HS-AFM as a powerful tool in structural biology and molecular biophysics. We reason that the progress of AFM must comprise two axes: First, making AFM/HS-AFM a better tool. With the above-mentioned methods, we made significant progress in this axis over the last ∼5 years. Second, making AFM/HS-AFM data an integrated part of the structural biology / molecular biophysics toolbox. To this end, we are developing now methods and data analysis and interpretation methods that interface with cryo-EM and X-ray crystallography, where AFM/HS-AFM can contribute the urgently needed information about structure, dynamics and conformations under close-to-native conditions.