title: Three-dimensional structure determination protocol for noncrystalline biomolecules using x-ray free-electron laser diffraction imaging.
abstract: Coherent and intense x-ray pulses generated by x-ray free-electron laser (XFEL) sources are paving the way for structural determination of noncrystalline biomolecules. However, due to the small scattering cross section of electrons for x rays, the available incident x-ray intensity of XFEL sources, which is currently in the range of 10(12)-10(13) photons/μm(2)/pulse, is lower than that necessary to perform single-molecule diffraction experiments for noncrystalline biomolecules even with the molecular masses of megadalton and submicrometer dimensions. Here, we propose an experimental protocol and analysis method for visualizing the structure of those biomolecules by the combined application of coherent x-ray diffraction imaging and three-dimensional reconstruction methods. To compensate the small scattering cross section of biomolecules, in our protocol, a thin vitreous ice plate containing several hundred biomolecules/μm(2) is used as sample, a setup similar to that utilized by single-molecule cryoelectron microscopy. The scattering cross section of such an ice plate is far larger than that of a single particle. The images of biomolecules contained within irradiated areas are then retrieved from each diffraction pattern, and finally provide the three-dimensional electron density model. A realistic atomic simulation using large-scale computations proposed that the three-dimensional structure determination of the 50S ribosomal subunit embedded in a vitreous ice plate is possible at a resolution of 0.8 nm when an x-ray beam of 10(16) photons/500×500 nm(2)/pulse is available.
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Tomotaka
authors.initials: T
authors.lastname: Oroguchi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Nakasako
mesh_terms: D001704:Biopolymers / Q000737:chemistry*; D003460:Crystallization; D007834:Lasers; D008968:Molecular Conformation; D014961:X-Ray Diffraction / Q000379:methods*
chemical_list: D001704:Biopolymers