TY - CHAP
T1 - Cryo-Electron Tomography of Reconstituted Biomolecular Condensates
AU - Tollervey, Fergus
AU - Zhang, Xiaojie
AU - Bose, Mainak
AU - Sachweh, Jenny
AU - Woodruff, Jeffrey B.
AU - Franzmann, Titus M.
AU - Mahamid, Julia
N1 - Funding Information:
We thank members of the Mahamid and Woodruff groups, the Electron Microscopy Core Facility and the EMBL cryo-EM platform, for invaluable input and support. M.B. was supported by a fellowship from the EMBL Interdisciplinary Postdoctoral Program (EI3POD) under Marie Skłodowska-Curie Actions COFUND (664726). J.M. acknowledges funding from the EMBL and the European Research Council (ERC 3DCellPhase-760067).
Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - 3D tomograms
KW - Molecular architecture
KW - Phase diagrams
KW - Phase separation
KW - Plunge freezing
KW - Protein/RNA condensates
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U2 - 10.1007/978-1-0716-2663-4_15
DO - 10.1007/978-1-0716-2663-4_15
M3 - Chapter
C2 - 36227480
AN - SCOPUS:85139880621
T3 - Methods in Molecular Biology
SP - 297
EP - 324
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -