Single particle cryo-EM reconstruction of 52\u2009kDa streptavidin at 3.2 Angstrom resolution

Xiao Fan,Xing Zhang,Jin-Can Zhang,Jia Wang,Hong-Wei Wang,Hai-Lin Peng,Jianlin Lei,Lingyun Zhao,Zi Yang

doi:10.1038/s41467-019-10368-w

Xiao Fan, Xing Zhang + Show 7 more

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https://doi.org/10.1038/s41467-019-10368-w

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Abstract

The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa.

Highlights

With the recent technical breakthroughs, cryo-EM has rapidly become one of the most powerful and efficient technology to investigate structures of macromolecules at near atomic resolution
As single particle analysis (SPA) cryo-EM has become a powerful method in solving many supramolecular complexes with large molecular weight, people keep wondering how small a molecule can be solved at near atomic resolution by this method
We demonstrated that using volta phase plate (VPP) and Cs-corrector, SPA cryo-EM can solve SA with molecular weight of about 50 kDa at ~3 Å resolution, good enough to determine the ligand binding site

Summary

Introduction

With the recent technical breakthroughs, cryo-EM has rapidly become one of the most powerful and efficient technology to investigate structures of macromolecules at near atomic resolution. New algorithms based on Bayesian statistics have greatly improved the efficiency of extracting signals from noisy micrographs and heterogeneous datasets[5,6,7,8,9] Nowadays, it has become more and more routine to get the reconstruction of a well behaved protein with molecular weight larger than 300 kDa at ~3 Å resolution. The major hurdle lies in the weak contrast of small-sized molecules embedded in vitreous ice under the conventional transmission electron microscopy (CTEM) Another major obstacle remaining in SPA cryoEM is the adsorption of proteins to the air-water interface of the thin layer of solution during the cryo-specimen preparation[3,10,11]. The smallest protein solved by CTEM using SPA at near atomic resolution is the 3.8 Å resolution structure of the 93 kDa isocitrate dehydrogenase (IDH)[12]

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