Alternatives for Uranyl Acetate Negative Staining With Respect to the Resolution Obtained After Single Particle Analysis of Erythrocruorin

Although cryo-electron microscopy is widely used for determining high-resolution structures, negative staining of proteins and supramolecular aggregates remain a valuable screening method, especially for low-abundant proteins. The most commonly used contrasting agent is uranyl acetate (UAc). However, due to its toxicity and the strong regulations connected to the use of uranium compounds, there is an ongoing search for alternative stains, with the focus being shifted to lanthanide and tungsten compounds. In this study, the performance of neodymium acetate (NdAc), europium acetate (EuAc), ytterbium acetate (YbAc), phosphotungstic acid, and the commercially available UA-Zero as negative staining agents for single molecule electron microscopy is evaluated. We focus not only on the observed contrast but also on the resolution that can be obtained after negative staining and single-particle analysis. The findings demonstrate that NdAc, EuAc, and UA-Zero can be good alternatives for the overall superior UAc. Additionally, we explore the working pH range of the stains and discuss the usability of the compounds in terms of availability and regulations.