Novel Integrative Protocol for Reconstructing Large Macromolecules Using Atomic Force Microscopy Data and Computational Docking

The drastic reduction of novel folds in proteins newly determined by x-ray crystallography suggests that large macromolecules are built from domains with already known structures. We have developed a novel integrative protocol that combines experimentally-measured topographic surfaces of single molecules with atomic coordinates of molecular constituents of large proteins or assemblies. Topographic surfaces are obtained using high-resolution atomic force microscopy (AFM) imaging. The present integrative method is based on real-space docking of macromolecular constituents beneath the experimental topographic surface. Assembly of molecular constituents is performed using a combinatorial approach. Only steric clashes between assembled constituents are computed; assemblies having more than a given threshold of bumps are eliminated. The goodness of fit is obtained by a score named E-factor which determines the agreement between the experimental topographic surface with that of the assembled constituents. A proof of concept has been determined on three different systems: Immunoglobulin G, Tobacco mosaic virus, and Aquaporin Z. Results demonstrated that partial topographic surface is adequate for complete macromolecular reconstruction. This protocol may be extremely useful for "difficult proteins" such as membrane proteins, partially unfolded proteins, and hard-to-produce proteins.