Four-dimensional imaging for cryo-electron microscopy experiments using molecular simulations and manifold learning.

Elucidating protein conformational changes is essential because conformational changes are closely related to the functions of proteins. Cryo-electron microscopy (cryo-EM) experiment can be used to reconstruct protein conformational changes via a method that involves using the experimental data (two-dimensional protein images). In this study, a reconstruction method, referred to as the "four-dimensional imaging," was proposed. In our four-dimensional imaging technique, the protein conformational change was obtained using the two-dimensional protein images (the three-dimensional electron density maps used in previously proposed techniques were not used). The protein conformation for each two-dimensional protein image was obtained using our original protocol with molecular dynamics simulations. Using a manifold-learning technique and two-dimensional protein images, the protein conformations were arranged according to the conformational change of the protein. By arranging the protein conformations according to the arrangement of the protein images, four-dimensional imaging is constructed. A simulation for a cryo-EM experiment demonstrated the validity of our four-dimensional imaging technique.