Structure Determination by Single-Particle Cryo-Electron Microscopy: Only the Sky (and Intrinsic Disorder) is the Limit.

Emeka Nwanochie,Vladimir N Uversky

doi:10.3390/ijms20174186

Abstract

Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by these powerful techniques. Despite their wide utilization in protein structure determination, these two techniques have logical limitations, with X-ray crystallography being unsuitable for the analysis of highly dynamic structures and with NMR spectroscopy being restricted to the analysis of relatively small proteins. In recent years, we have witnessed an explosive development of the techniques based on Cryo-electron microscopy (Cryo-EM) for structural characterization of biological molecules. In fact, single-particle Cryo-EM is a special niche as it is a technique of choice for the structural analysis of large, structurally heterogeneous, and dynamic complexes. Here, sub-nanometer atomic resolution can be achieved (i.e., resolution below 10 Å) via single-particle imaging of non-crystalline specimens, with accurate 3D reconstruction being generated based on the computational averaging of multiple 2D projection images of the same particle that was frozen rapidly in solution. We provide here a brief overview of single-particle Cryo-EM and show how Cryo-EM has revolutionized structural investigations of membrane proteins. We also show that the presence of intrinsically disordered or flexible regions in a target protein represents one of the major limitations of this promising technique.

Highlights

Brief Historic Overview of Methods of Structural BiologyWhen faced with a problem, a reduction to its component parts is generally a common practice in science
It is important to note that the relationship between protein structure and function is not always one of complete linearity, and this complexity of the structure–function relationship is commonly exemplified by the class of intrinsically disordered proteins and polypeptides [2,3,4,5,6,7,8]
The establishment of the technique as an investigative tool applicable across disciplines took approximately half a century [10]. Both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy were adopted over time as primary methods for structure determination at atomic resolution

Summary

Introduction

When faced with a problem, a reduction to its component parts is generally a common practice in science. The establishment of the technique as an investigative tool applicable across disciplines took approximately half a century [10] Both X-ray crystallography and NMR spectroscopy were adopted over time as primary methods for structure determination at atomic resolution. Sci. 2019, 20, 4186 notable developments is the utilization of a transverse relaxation optimized spectroscopy (TROSY) This is a technique for suppression of transverse relaxation in multidimensional NMR experiments [31] combined with specific labeling schemes [32]; the use of selective protonation of methyl groups in highly deuterated background [33], incorporation of (1H-δ methyl)-leucine and (1H- γ methyl)-valine into 15N-, 13C-, 2H-labeled proteins [34], or using reductive 13C-methylation of lysines [35]. The last 5 years witnessed a major leap in the number of yearly deposited PDB structures resolved by cryo-EM [39]

Cryo-EM

Specimen Preparation for Single Particle Cryo-EM Analysis

Data Acquisition and Image Processing

Single-Particle Cryo-EM Analysis of Membrane Proteins

Conclusions