High-Resolution Single-Molecule Structure Revealed by Electron Microscopy and Individual Particle Electron Tomography.

Lei Zhang,Gang Ren

doi:10.4172/2161-0398.1000e103

Abstract

The protein is naturally dynamic and heterogeneous in solution. Protein dynamics involves both equilibrium fluctuations that regulate biological function and other non-equilibrium effects of biological motors, which convert chemical energy to mechanical energy. However, a single, unique structure of protein determined from X-ray crystal and conventional single-particle electron microscopy is insufficient to encompass the dynamic nature of proteins in solution. Structure determination of dynamic and heterogeneous protein is essentially required the determination of each individual particle of protein. Recently, Drs. Gang Ren and Lei Zhan published the first single molecule three-dimensional (3D) EM images of individual proteins ever obtained with enough clarity to determine their structure, an IgG antibody (14 Å resolution) and a 17nm HDL (36 Å resolution). These results depended upon four innovations: i) improved cryo-electron microscopy (cryoEM) sample preparation and Electron microscopy (EM) operation conditions resulted in the successful imaging of a 17 nm HDL particle (120-200kDa) by cryo-electron tomography (cryoET); ii) developed an optimized NS (OpNS) protocol that eliminates the rouleau artifact that has plagued EM research for three decades. This OpNS protocol provides high-contrast single lipoprotein images with similar size (<5%) and shape (<5%) to that seen by cryoEM; iii) developed a high-resolution and high contrast sample preparation protocol, cryo-positive-staining (cryoPS) that allows direct visualization of the secondary structure of a small protein, such as the β-strands in CETP and the helical double belt of apoA-I in spherical HDL; iv) developed a robust tomography reconstruction method, Individual Particle Electron Tomography (IPET) that is a high-resolution, high throughput reconstruction method that, to the best of our knowledge, is the only method for determining an individual protein structure. Remarkably, IPET went against the conventional wisdom that a single protein can NOT be reconstructed by EM and this opens a door for the study of protein dynamics via a particle-by-particle structural comparison.

Highlights

A single, unique structure of protein determined by X-ray crystal is often used for studies in structure–function relationships
Other than a theoretical calculation approach, such as molecular dynamics simulation is able to link structure and dynamics by enabling the exploration of the conformational energy landscape accessible to protein molecules [1,2], there is no experimental approach to determine the dynamic structure at atomic-resolution level because current structure determination techniques, including X-ray crystallography and single-particle electron microscopy, require an averaging of the signals from thousands to millions different particles
A fundamental solution for structure determination of dynamics protein is to determine the structure of each individual particle of protein

Summary

Introduction

A single, unique structure of protein determined by X-ray crystal is often used for studies in structure–function relationships. Optimization of Cryo-Electron Tomography (cryoET) Technique for High-Resolution Imaging of small Protein under a Near Native State

Results

Conclusion