Three-Dimensional Reconstruction of Trypanosoma brucei Editosomes Using Single-Particle Electron Microscopy
RNA editing within the mitochondria of kinetoplastid protozoa is performed by a multicomponent macromolecular machine known as the editosome. Editosomes are high molecular mass protein assemblies that consist of about 15–25 individual polypeptides. They bind pre-edited transcripts and convert them into translation-competent mRNAs through a biochemical reaction cycle of enzyme-catalyzed steps. At steady-state conditions, several distinct complexes can be purified from mitochondrial detergent lysates. They likely represent RNA editing complexes at different assembly stages or at different functional stages of the processing reaction. Due to their low cellular abundance, single-particle electron microscopy (EM) represents the method of choice for their structural characterization. This chapter describes a set of techniques suitable for the purification and structural characterization of RNA editing complexes by single-particle EM. The RNA editing complexes are isolated from the endogenous pool of mitochondrial complexes by tandem-affinity purification (TAP). Since the TAP procedure results in the isolation of a mixture of different RNA editing complexes, the isolates are further subjected to an isokinetic ultracentrifugation step to separate the complexes based on their sedimentation behavior. The use of the “GraFix” protocol is presented that combines mild chemical cross-linking with ultracentrifugation. Different sample preparation protocols including negative staining, cryo-negative staining, and unstained cryotechniques as well as the single-particle image processing of electron microscopical images are described.
Key wordsRNA editing Editosome Trypanosoma brucei Tandem-affinity purification (TAP) GraFix Surface plasmon resonance (SPR) Density gradient centrifugation Electron microscopy (EM) Cryo-EM Single-particle image processing
MMG and BS are supported by a grant from the Danish Center for Scientific Computing (DCSC). HS is supported by a grant of the Bundesministerium für Bildung und Forschung (BMBF) and a European “3D Repertoire” grant. HUG is supported as an International Scholar of the Howard Hughes Medical Institute (HHMI) and by the German Research Foundation (DFG).
- 9.Kastner, B., Fischer, N., Golas, M. M., Sander, B., Dube, P., Boehringer, D., Hartmuth, K., Deckert, J., Hauer, F., Wolf, E., Uchtenhagen, H., Urlaub, H., Herzog, F., Peters, J. M., Poerschke, D., Lührmann, R., and Stark, H. (2008) GraFix: sample preparation for single-particle electron cryomicroscopy Nat Methods 5, 53–5.PubMedCrossRefGoogle Scholar
- 26.Chiu, W., Downing, K. H., Dubochet, J., Glaeser, R. M., Heide, H. G., Knapek, E., Kopf, D. A., Lamvik, M. K., Lepault, J., Robertson, J. D., Zeitler, E., and Zemlin, F. (1986) Cryoprotection in electron microscopy J Microsc 141, 385–91.Google Scholar
- 40.Migneault, I., Dartiguenave, C., Bertrand, M. J., and Waldron, K. C. (2004) Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking Biotechniques 37, 790–6, 8–802.Google Scholar