Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii

Kelley, R.; Khavnekar, S.; Righetto, R. D.; Heebner, J.; Obr, M.; Zhang, X.; Chakraborty, S.; Tagiltsev, G.; Michael, A. K.; van Dorst, S.; Waltz, F.; McCafferty, C. L.; Lamm, L.; Zufferey, S.; Van der Stappen, P.; van den Hoek, H.; Wietrzynski, W.; Harar, P.; Wan, W.; Briggs, J. A. G.; Plitzko, J. M.; Engel, B. D.; Kotecha, A.

doi:10.1101/2024.12.28.630444

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bioRxiv · 10.1101/2024.12.28.630444

Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii

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Abstract

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii, a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to [~]200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

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Kelley, R., Khavnekar, S., Righetto, R. D., Heebner, J., Obr, M., Zhang, X., Chakraborty, S., Tagiltsev, G., Michael, A. K., van Dorst, S., Waltz, F., McCafferty, C. L., Lamm, L., Zufferey, S., Van der Stappen, P., van den Hoek, H., Wietrzynski, W., Harar, P., Wan, W., Briggs, J. A. G., Plitzko, J. M., Engel, B. D., Kotecha, A.. 2024-12-28. Towards community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii. https://doi.org/10.1101/2024.12.28.630444

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