Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Mickaël V Cherrier,Daphna Fenel,Emmanuelle Neumann,Yvain Nicolet,Benoit Arragain,Xavier Vernède,Guy Schoehn,Lydie Martin,Juan C Fontecilla-Camps

doi:10.3390/biom12030441

Abstract

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.

Highlights

Cryo-Electron Microscopy.Many d-block elements of the periodic table, known as transition metals, are essential to all living organisms
The successive vacuum/flushing steps needed to remove all traces of oxygen would cause massive evaporation and partial loss of the cryogenic fluids contained in the cryo-electron microscopy (cryo-EM) grid flash-cooling
We took advantage of the protocol already developed in our laboratory to successfully flash-cool metalloprotein crystals under anaerobic conditions inside a glovebox [20]

Summary

Introduction

Many d-block elements of the periodic table, known as transition metals, are essential to all living organisms They confer unique properties to proteins, thereby extending the repertoire of possible chemical reactions well beyond those made possible by amino acids alone. A bioinformatic study comparing the genome of many organisms from the different kingdoms of life determined that approximately one third of all protein activities depends on transition metals [1]. Their importance in energy metabolism and the observed similarity between several organometallic cofactors and minerals led to the hypothesis that mineral surfaces played a key role in the emergence of life on Earth [2]

Methods

Results

Conclusion