Dynamic personalities and structural heterogeneities of proteins are essential for understanding their proper functions. However,structure determination of dynamic/heterogeneous protein is limited by current technologies, such as X-ray crystallography and electron microscopy (EM) single particle analysis, both of which generally require averaging from thousands of different proteins based on an assumption that these thousand proteins are structurally identical. Electron tomography (ET) provides a tool for visualization of a unique biological object from a series of tilted viewing angles. Conventional reconstruction methods using whole micrographs provide tools for 3-dimensional (3D) reconstructions of a large biological object, such as bacteria, and sections of cell. However, for small and low-symmetry proteins, these methods have limited power in reconstruction resolution. Recently, Ren’s group reported a so-called individual-particle electron tomography (IPET) method, in which, a “focused electron tomography reconstruction (FETR)” algorithm was proposed to improve the reconstruction resolution by decreasing the image size so that it only contains a single-instance protein. IPET method requires no pre-given initial model or average of multiple molecules, but also can tolerate certain levels of measuring tilt errors. In this review, we demonstrate the IPET/FETR method in detail to share this current progress with the researchers in China. We believed IPET is a new and robust approach to determine the structure of a single/individual molecule that is a basis for studying the dynamic character and structural heterogeneity of macromolecule via comparison and structural analyses of structures determined from different individual macromolecules. Contents
1 Introduction
2 IPET method and FETR algorithm
2.1 Generating simulated cryoET data
2.2 Basic tools for image processing
2.3 Focused electron tomography reconstruction (FETR) algorithm
3 Validation of focused ET reconstruction method by real experimental data
3.1 3D reconstruction of a human IgG antibody by negative-staining ET
3.2 3D reconstruction of a high-density lipoprotein by cryoET
4 Conclusion and outlook