3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography.

Lei Zhang,Matthew J. Rames,Shengli Zhang,Xing Zhang,Huimin Tong,Gang Ren,Bo Peng

doi:10.1038/srep09803

Abstract

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Highlights

Understanding how proteins function in isolation and in their native context requires merging several molecular-level techniques that explore the interplay of protein structure and dynamics[1]
Transmission electron microscopy (TEM) serves as a tool for individual protein imaging at atomic resolution, while electron tomography (ET) images an individual protein particle from a series of tilting angles
We reported a method for 3D reconstruction of an individual protein particle, named individual-particle electron tomography (IPET) reconstruction[12]

Summary

Introduction

Understanding how proteins function in isolation and in their native context requires merging several molecular-level techniques that explore the interplay of protein structure and dynamics[1]. Current structural determination tools such as X-ray crystallography and single-particle reconstructions often reveal a single unique structure in which protein conformational flexibilities and dynamics are often absent. This is a result of the averaging process, in which thousands to millions of protein molecules assumed to share a single conformation are averaged together in order to enhance signal from proteins and to achieve a common structure. We applied this method and reconstructed a few 3D structures at an intermediate resolution (~1–4 nm) from both negative-staining and cryo-electron microscopy samples[10,12]. The distribution of domain locations and orientation of conformations provided the basis for statistical analysis of antibody flexibility and dynamics

Methods

Results

Conclusion