Lutter, L.; Beal, D. M.; Stanley, M.; Roobol, J.; Martin, S.; Budge, J. D.; Lee, P. E.; Nemoto-Smith, E.; Brown, I.; Warren, M. J.; Smales, C. M.; Xue, W.-F.

Atomic force microscopy (AFM) is a versatile multi-modal imaging method frequently used for structural characterisation of biological surfaces at the nanoscale. However, AFM-based three-dimensional single-particle reconstruction has hitherto not been possible due to the tip-sample convolution artifact that distorts AFM images of individual molecules, and the disconnect between two-dimensional AFM images of surface deposited molecules and their three-dimensional structures. Here, three-dimensional single-particle analysis was developed for rapid structure-based validation of protein structures using as few as a single AFM topology image, based on contact-point reconstruction AFM (CPR-AFM) in an integrative approach with cryo-electron microscopy maps available in the Electron Microscopy Data Bank by template matching. This approach was demonstrated on the structural validation of recombinant trimeric ectodomain of SARS-CoV-2 Spike glycoprotein to show its immediate utility as a rapid structure-based sample quality control method in the recombinant expression and purification of the Spike protein samples that can be used in vaccines and therapeutics research. These results show that three-dimensional single-particle reconstruction by AFM is possible, that high signal-to-noise AFM imaging offers a rapid and cost-effective way of validation or identification of three-dimensional protein structures at single particle level, and that AFM can be linked to structural data derived from methods such as cryo-electron microscopy, resulting in integrative methodologies with new capabilities for structural biology.