Asymmetric Small Protein Structure Determination by Individual Particle Electron Tomography

Abstract

Single-particle reconstruction of electron microscopy has been successfully used to determine high-resolution structure of proteins that are highly symmetric with a large macromolecular complex (molecular weight, MW > 200 kDa). However, for asymmetric small proteins (MW < 100 kDa), this approach still faces challenges to obtain intermediate resolution structure due to difficulties attaining a reliable initial model. To obtain the initial model, especially from an asymmetric small protein, we present a novel method by individual particle electron tomography (IPET). In this method, an individual protein particle was targeted and imaged from a serials of tilt-angles by electron tomography (ET) using our reported focused ET reconstruction (FETR) algorithm to reconstruct the three-dimensional (3D) structure from those tilt serial of images that were acquired from each instance of the molecule. To demonstrate the method, we imaged and reconstructed structure of small protein, cholesteryl ester transfer protein (CETP), whose molecular weight is only 53 kDa. The experimental sample was prepared by an optimized negative-staining protocol and was acquired under the near Scherzer-focus imaging condition. To investigate the reliability of the 3D density map reconstructed from single-instance particle, we statistically analyzed the map by comparing it with the crystal structure and the density map refined from this initial model, followed by the single-particle refinement procedure. Although Fourier shell cross-correlation (FSC) analysis shows the resolution of the tomographic reconstruction and single-particle reconstruction are both around 15 A. These results suggest this approach can be used to obtain the initial model for single-particle reconstruction, moreover, the similar quality and resolution obtained from the IPET and single-particle reconstruction suggest that our reported IPET and FETR reconstruction can be used directly to determine the single-instance molecule structure at intermediate resolution (1-2 nm).