Conformational analysis of the structure of ribosome fit into electron microscopy density maps with normal mode analyses and molecular dynamics simulations

Ishida, Hisashi; Matsumoto, Atsushi; Tsutsumi, Yu*; Yura, Kei

We are developing an EM density-fitting refinement method to improve the modeled 3D structure of supra-biomolecules by alleviating the steric stress of atoms while retaining the condition that the constituent molecules fit in the EM density map. The method was applied to ribosome. We used an atomic structure of Thermus thermophilus 70S ribosome which was determined by X-ray crystallography (PDB code: 1YL3 and 1YL4). The target EM data from Escherichia coli 70S ribosome, which were observed under various reaction conditions from the initial to the final stage of the translation of messenger RNA, were retrieved from the electron microscopy database at the European Bioinformatics Institute. In our method, the X-ray structure of ribosome was fit into the EM maps as a rigid-body by matching the three inertial axes of the X-ray structure with those of each EM map first. Then, the atomic coordinates were transformed along the directions of low frequency modes calculated by NMA until the best fit was achieved. Finally, MD simulations in which ribosomal RNAs were treated as flexible molecules and the other molecules were treated as rigid-body molecules were performed to produce a refined 3D structure of ribosome without atomic collisions. The MD simulations also optimized the match between the modeled 3D structures and the EM maps. It was found that the NMA simulations could match the 3D structure and the EM maps in the range of 59-78%, while the MD simulation matched them in the range of 56-82%. In the MD simulation, the center of masses and orientations of constituent molecules of ribosome in the X-ray structure and the refined structure were compared, and the amplitudes of the differences of the centers of masses and the orientations was about 2 Å and about 4º respectively on average. It is considered that the structural changes indicate the functional movements in ribosome under various reaction conditions.