Purification of ribosomes from human embryonic stem (hES) cells for high-resolution Cryo-EM structural studies.

Abstract

Human ribosome structures at near-atomic resolution have been determined predominantly from transformed cell lines, often using translation inhibitors during purification that can introduce structural artifacts. We present a robust and scalable protocol for purifying 80S ribosomes from human embryonic stem (hES) cell cultures. The method addresses two common bottlenecks for structural studies of ribosomes from pluripotent cells: limited starting material and structural perturbations introduced by translation inhibitors such as cycloheximide or anisomycin. By combining gentle lysis and rapid clarification with a sucrose-cushion concentration step followed by gradient based purification the workflow preserves native ribosome conformations and yields highly pure 80S ribosomes suitable for single-particle cryo-EM at near-atomic resolution model building. The workflow was reproduced across independent preparations with consistent yields and map quality. As expected for inhibitor-free purification, no density corresponding to elongation inhibitors was observed in ribosomal functional centers. We supply a detailed, scalable protocol that includes buffer recipes, expected yields, quality control and troubleshooting steps, and recommendations for cryo-EM grid preparation. The procedure is transferable to other sensitive cell types (for example, primary B cells and iPS cells). This workflow expands access to native human ribosomes from non-transformed, developmentally relevant cells for structural and mechanistic studies of translation.