Structural Determination of Heterogeneous Protein by Individual-Particle Electron Tomography - Combination of Electron Tomography and Local Refinement Reconstruction Method for High-Resolution Structural Determination of Each Individual Protein Particle

Abstract

The dynamic nature and structural heterogeneity of proteins is essential to their function. However, the purification of these proteins, such as lipoproteins, liposomes, and antibodies, is difficult or even impossible. Heterogeneity prevents structural determination by current technologies, such as X-ray crystallization, NMR, and even single-particle electron cryo-microscopy (cryo-EM). Here, we report a method to determine the structure of heterogeneous proteins by determining the structure of individual protein particle. This method, which we call individual-particle electron tomography (IPET), is the combination of current electron tomography (ET) technique with our reconstruction program. In brief, we used ET to image each individual protein particle from a series of tilt angles ranging from −70° to +70°. Then, we reconstructed the 3D density map from each individual particle's images by searching each image's global center within the particle local area via an iteration and refinement algorithm and a set of dynamic filters and maskers. This method was particularly effective in processing the highly noisy and low-contrast cryo-EM images for generating the high resolution 3D density map. To elucidate our methodology and demonstrate its capabilities, we used a set of simulated cryo-EM data, a tilt series of images based on an individual transporter (∼100kDa). The reconstructed 3D density map contains fine structural details, such as α-helices, at high resolution (beyond 10Å indicated by the Fourier shell correlation analysis). This program works robustly on both simulated data and real data (see our other abstracts). Considering IPET is the only method for high resolution structural determination based on an individual object of protein, we propose it as a novel method to study the structure of highly dynamic and heterogeneous proteins.