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Ishida, Hisashi; Matsumoto, Atsushi; Tsutsumi, Yu*; Yura, Kei
Proceedings of 16th International Microscopy Congress (IMC 2006), P. 242, 2006/09
Supra-biomolecules, which contain numerous proteins and nucleic acids, function when the constituent molecules are assembled. Therefore, it is important to determine not only the 3D structures of the constituent molecules but also the 3D structure of the supra-biomolecule. Although X-ray crystallography can determine the atomic coordinates of biomolecules, it has difficulty in handling supra-biomolecules, because crystals of huge molecules cannot be made with ease. Single particle analysis using an electron microscope (EM) has been used to observe the structure of supra-biomolecules, but the resolution of the EM image has only achieve to the atomic level in a limited situation. Therefore, several attempts have been carried out to determine the 3D structure of supra-biomolecules in atomic resolution by fitting the constituent molecules, determined by X-ray crystallography, into an EM density map. In those attempts, each constituent molecule is usually fit into the EM density map manually, and the constituent molecules may have atomic collisions at their interfaces.
Ishida, Hisashi; Matsumoto, Atsushi; Tsutsumi, Yu*; Yura, Kei
no journal, ,
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.
Tsutsumi, Yu*; Matsumoto, Atsushi; Yura, Kei; Ishida, Hisashi
no journal, ,
We are developing an EM density-fitting refinement method to improve the modeled 3D structure of supramolecules. Our method first uses rigid body fitting to carry out initial fitting, and then uses a molecular dynamic simulation, in which a target function together with the standard all-atom energy function is used to constrain the atomic structure into the EM map. Here we report the application of this method to the Thermus thermophilus 70S ribosome. In this study we have fit the X-ray crystallographic structure of the ribosome into multiple EM images which show the ribosome in different stages of translation. The refined structures are compared and as a result differences in structures are observed. The differences are thought to indicate the functional movements of the ribosome.
Ishida, Hisashi; Tsutsumi, Yu*; Matsumoto, Atsushi; Yura, Kei
no journal, ,
We are developing an EM density-fitting refinement method to improve the modeled 3D structure of supramolecules. Our method first uses rigid body fitting to carry out initial fitting, and then uses a molecular dynamic simulation, in which a target function together with the standard all-atom energy function is used to constrain the atomic structure into the EM map. Here we report the application of this method to the Thermus thermophilus 70S ribosome. In this study we have fit the X-ray crystallographic structure of the ribosome into multiple EM images which show the ribosome in different stages of translation. The refined structures are compared and as a result differences in structures are observed. The differences are thought to indicate the functional movements of the ribosome.
Ishida, Hisashi; Matsumoto, Atsushi; Tsutsumi, Yu*; Yura, Kei
no journal, ,
no abstracts in English
Ishida, Hisashi; Matsumoto, Atsushi; Tsutsumi, Yu*; Yura, Kei
no journal, ,
We developed 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. Our method first uses rigid body fitting to carry out initial fitting and then uses a molecular dynamics (MD) simulation. It was found that (1) the refinement could fit the atomic structure of ribosome into the EM density maps with scores ranging from 68.5 to 83.1%, (2) the movement of the head of the 30S subunit largely contributed to the structural change of the bridge between 30S and 50S ribosome, (3) the bridges in helix 44 of 16S rRNA were well conserved throughout all the structures, (4) there were, at least, five exit tunnels with a diameter more than six angstroms, and (5) the structure of a tunnel was dynamic and its entrance was closed at the initial stage of protein synthesis.
Ishida, Hisashi; Tsutsumi, Yu*; Matsumoto, Atsushi; Yura, Kei
no journal, ,
no abstracts in English
Ishida, Hisashi; Tsutsumi, Yu*; Matsumoto, Atsushi; Yura, Kei
no journal, ,
no abstracts in English