Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent technology advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including less amount of protein required and avoiding protein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.