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. 2023:2563:297-324.
doi: 10.1007/978-1-0716-2663-4_15.

Cryo-Electron Tomography of Reconstituted Biomolecular Condensates

Fergus Tollervey  1   2 , Xiaojie Zhang  1 , Mainak Bose  3 , Jenny Sachweh  1   4 , Jeffrey B Woodruff  5 , Titus M Franzmann  6 , Julia Mahamid  7
Affiliations

Cryo-Electron Tomography of Reconstituted Biomolecular Condensates

Fergus Tollervey et al. Methods Mol Biol. 2023.

Abstract

The assembly of membraneless compartments by phase separation has recently been recognized as a mechanism for spatial and temporal organization of biomolecules within the cell. The functions of such mesoscale assemblies, termed biomolecular condensates, depend on networks of multivalent interactions between proteins, their structured and disordered domains, and commonly also include nucleic acids. Cryo-electron tomography is an ideal tool to investigate the three-dimensional architecture of such pleomorphic interaction networks at nanometer resolution and thus form inferences about function. However, preparation of suitable cryo-electron microscopy samples of condensates may be prone to protein denaturation, low retention of material on the sample carrier, and contamination associated with cryo-sample preparation and transfers. Here, we describe a series of protocols designed to obtain high-quality cryo-electron tomography data of biomolecular condensates reconstituted in vitro. These include critical screening by light microscopy, cryo-fixation by plunge freezing, sample loading into an electron microscope operated at liquid nitrogen temperature, data collection, processing of the data into three-dimensional tomograms, and their interpretation.

Keywords: 3D tomograms; Molecular architecture; Phase diagrams; Phase separation; Plunge freezing; Protein/RNA condensates.

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