Isolation of Disease-relevant p53 for Cryo-Electron Microscopy Analysis

Abstract Tumor suppressor protein TP53 (p53) plays a multi-faceted role in all cells of thehuman body. Sadly, mutations in the TP53 gene are involved in nearly ~50% of tumors,spurring erratic cell growth and disease progression. Until recently, structural informationfor p53 remained incomplete and there are limited studies on native p53 produced inhuman tumors. Here, we present a highly reproducible and effective protocol to extract,enrich, and purify native p53 protein assemblies from cancer cells for downstreamstructural studies. This method does not introduce purification tags into the p53 gene andmaintains naturally occurring modifications. In conjunction with cryo-Electron Microscopytechniques, we determined new structures for p53 monomers (~50 kDa) and tetramers(~200 kDa) at spatial resolutions of ~4.8 Å and ~7 Å, respectively.1 These modelsrevealed new insights for flexible regions of p53 along with biologically-relevantubiquitination sites. Combining biochemical and structural imaging protocols, we aim tobuild a better understanding of native p53’s impact in cancer formation.