Exploring the molecular landscape of Chlamydomonas with in situ cryo-electron tomography
Benjamin D. Engel1), Miroslava Schaffer1), Sahradha Albert1), Luis Kuhn Cuellar1), Stefan Pfeffer1), Jürgen M. Plitzko1), Wolfgang Baumeister1)
1) Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Germany (firstname.lastname@example.org)
We are leveraging new advances in cryo-electron tomography (cryo-ET) to investigate macromolecular complexes within the native cellular environment. Thin slices of vitrified Chlamydomonas reinhardtii cells are prepared by cryo-focused ion beam (cryo-FIB) milling and then imaged by state-of-the-art cryo-ET. The resulting 3D views of cellular volumes have provided new insights into the molecular organization of organelles. Within the chloroplast, we identified fine membrane tubules that likely serve as conduits for the directed diffusion of metabolites between the pyrenoid and the chloroplast stroma. We were also able to directly visualize photosynthetic complexes within native thylakoid membranes, as well as the organization of RuBisCO complexes in the pyrenoid. In the Golgi, we identified protein arrays that establish the narrow membrane spacing of the trans-Golgi cisternae, and also solved the in situ structure of the COPI coat complex. Subtomogram analysis of ER-bound ribosomes enabled us to dissect the molecular architecture of the TRAP translocon component. Current efforts include the structural characterization of proteasomes that encircle nuclear pore complexes on the inside of the nuclear envelope. In addition to the in situ characterization of individual macromolecular complexes, in the future we will aim for a visual proteomics approach to identify and classify every macromolecule within the cellular volumes.
Pfeffer S, Dudek J, Schaffer M, Ng BG, Albert S, Plitzko JM, Baumeister W, Zimmermann R, Freeze HH, Engel BD, Förster F. (2016). Dissecting the molecular organization of the translocon-associated protein complex. Nature Communications. In press.
Schaffer M, Mahamid J, Engel BD, Laugks T, Baumeister W, Plitzko J (2016). Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins. Journal of Structural Biology. Epub ahead of print, doi: 10.1016/j.jsb.2016.07.010.
Asano S, Engel BD, Baumeister W (2015). In situ cryo-electron tomography: a post-reductionist approach to structural biology. Journal of Molecular Biology 428: 332–343.
Engel BD, Schaffer M, Albert S, Asano S, Plitzko JM, Baumeister W (2015). In situ structural analysis of Golgi intracisternal protein arrays. Proceedings of the National Academy of Sciences USA 112: 11264-11269.
Engel BD, Schaffer M, Cuellar LK, Villa E, Plitzko JM, Baumeister W (2015). Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. eLife 4: e04889.