Single particle analysis of protein structure using graphene liquid cell

Abstract

Many scientists are interest in liquid environment, because almost physical, chemical and biological reactions occur in the liquid environment. However, since the transmission electron microscopy (TEM) obligatorily need ultra-high vacuum for observing the specimens, it is impossible to preserve and observe liquid conditions without evaporation of liquid. To overcome these limitation, the cryo-EM, which prepare frozen specimens, and liquid-EM, which encapsulate liquid using thin membrane, have been researched.

Especially, observing the biomolecules using TEM is appreciated with recent development of cryo-EM. However, frozen specimens represent only static snap shot of biomolecules. Therefore, liquid-EM is required to observe dynamic phenomena by adopting the electron transparency membranes. And the graphene, which has atomic thickness, is desirable membrane materials for observing biomolecules.

In this thesis, single particle analysis of protein structure using graphene liquid cell (GLC) is accomplished. First, the graphene surface treatment methods are introduced to improve the wettability of graphene and protein adsorption efficiency for reliable sample preparation. Also, protein particles, which are sandwiched between graphene layers, are directly observed by using GLC and 3D model of protein in the GLC is reconstructed by suing single particle analysis. Finally, reconstructed 3D model is compared with reference models for evaluating reconstructed model.