Karapanagioti Foteini, Cissé Nicolas, Atamas Anastasiia, Stetsenko Artem, Punter Christiaan Michiel, Zuidersma Erica, Stuart Marc C A
Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, the Netherlands.
Stratingh Institute for Chemistry, University of Groningen, Groningen, the Netherlands.
Microsc Res Tech. 2025 Sep;88(9):2381-2391. doi: 10.1002/jemt.24865. Epub 2025 Mar 31.
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.
尽管冷冻电子显微镜被广泛用于确定高分辨率结构，但蛋白质和超分子聚集体的负染色仍然是一种有价值的筛选方法，特别是对于低丰度蛋白质。最常用的造影剂是醋酸铀酰（UAc）。然而，由于其毒性以及与铀化合物使用相关的严格规定，人们一直在寻找替代染色剂，重点已转向镧系元素和钨化合物。在本研究中，评估了醋酸钕（NdAc）、醋酸铕（EuAc）、醋酸镱（YbAc）、磷钨酸以及市售的UA-Zero作为单分子电子显微镜负染色剂的性能。我们不仅关注观察到的对比度，还关注负染色和单颗粒分析后可获得的分辨率。研究结果表明，NdAc、EuAc和UA-Zero总体上可成为UAc的良好替代品。此外，我们还探索了染色剂的工作pH范围，并从可用性和规定方面讨论了这些化合物的实用性。
