Three-dimensional single-particle reconstruction by atomic force microscopy imaging and template matching allows rapid structural-based validation of recombinant SARS-CoV-2 Spike protein from a single topology image.

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 for rapid structure-based quality-control in the recombinant expression and purification of 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, 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 AFM linked to cryo-electron microscopy-derived structural data result in integrative methodologies with new capabilities for structural biology.