title: IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.
abstract: The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
authors.affiliation: Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D020285:Cryoelectron Microscopy / Q000379:methods*; D018360:Crystallography, X-Ray; D055032:Electron Microscope Tomography / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods; D046911:Macromolecular Substances / Q000032:analysis / Q000737:chemistry; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D046911:Macromolecular Substances