Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii
Kelley R, Khavnekar S, Righetto RD, Heebner J, Obr M, Zhang X, Chakraborty S, Tagiltsev G, Michael AK, Van Dorst S, Waltz F, Mccafferty CL, Lamm L, Zufferey S, Van Der Stappen P, Van Den Hoek H, Wietrzynski W, Harar P, Wan W, Briggs JAG, Plitzko JM, Engel BD, Kotecha A. Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii. Molecular Cell.
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Author
Kelley, Ron;
Khavnekar, Sagar;
Righetto, Ricardo D.;
Heebner, Jessica;
Obr, MartinISTA 
;
Zhang, Xianjun;
Chakraborty, Saikat;
Tagiltsev, Grigory;
Michael, Alicia K.ISTA ;
Van Dorst, Sofie;
Waltz, Florent;
Mccafferty, Caitlyn L.
All
;
Zhang, Xianjun;
Chakraborty, Saikat;
Tagiltsev, Grigory;
Michael, Alicia K.ISTA ;
Van Dorst, Sofie;
Waltz, Florent;
Mccafferty, Caitlyn L.
All
Department
Abstract
In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate the structures of biomolecules in their native context. However, challenges remain for the efficient production and sharing of large-scale cryo-ET datasets. Here, we combined cryogenic plasma-based focused ion beam (cryo-PFIB) milling with recent advances in cryo-ET acquisition and processing to generate a dataset of 1,829 annotated tomograms of the green alga Chlamydomonas reinhardtii, which we provide as a community resource to drive method development and inspire biological discovery. To assay data quality, we performed subtomogram averaging 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 to empower visual proteomics.
Publishing Year
Date Published
2025-12-19
Journal Title
Molecular Cell
Publisher
Elsevier
Acknowledgement
Calculations were performed at the Max Planck Institute of Biochemistry and the Raven Supercomputer of the Max Planck Computing and Data Facility (MPCDF) in Garching, Germany; at the sciCORE (http://scicore.unibas.ch/) scientific computing center at the University of Basel, Switzerland; and at Thermo Fisher Scientific, in Eindhoven, the Netherlands. This work was supported by Thermo Fisher Scientific. All lamella preparations and tilt-series collections used in this work were conducted at Thermo Fisher R&D facilities in Brno and Eindhoven, utilizing Arctis and Krios microscopes. This work was also supported by the ERC consolidator grant “cryOcean” (fulfilled by the Swiss State Secretariat for Education, Research and Innovation, M822.00045) as well as a Swiss Nanoscience Institute PhD school grant to B.D.E. and P.V.d.S., an EMBO long-term postdoctoral fellowship (ALTF-383-2022) to G.T., an SNSF Postdoctoral Fellowship (project 210561) to F.W., a Boehringer Ingelheim Fonds fellowship to L.L., and by the Max Planck Society to J.A.G.B. and J.M.P.
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Cite this
Kelley R, Khavnekar S, Righetto RD, et al. Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii. Molecular Cell. doi:10.1016/j.molcel.2025.11.029
Kelley, R., Khavnekar, S., Righetto, R. D., Heebner, J., Obr, M., Zhang, X., … Kotecha, A. (n.d.). Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii. Molecular Cell. Elsevier. https://doi.org/10.1016/j.molcel.2025.11.029
Kelley, Ron, Sagar Khavnekar, Ricardo D. Righetto, Jessica Heebner, Martin Obr, Xianjun Zhang, Saikat Chakraborty, et al. “Toward Community-Driven Visual Proteomics with Large-Scale Cryo-Electron Tomography of Chlamydomonas Reinhardtii.” Molecular Cell. Elsevier, n.d. https://doi.org/10.1016/j.molcel.2025.11.029.
R. Kelley et al., “Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii,” Molecular Cell. Elsevier.
Kelley R, Khavnekar S, Righetto RD, Heebner J, Obr M, Zhang X, Chakraborty S, Tagiltsev G, Michael AK, Van Dorst S, Waltz F, Mccafferty CL, Lamm L, Zufferey S, Van Der Stappen P, Van Den Hoek H, Wietrzynski W, Harar P, Wan W, Briggs JAG, Plitzko JM, Engel BD, Kotecha A. Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii. Molecular Cell.
Kelley, Ron, et al. “Toward Community-Driven Visual Proteomics with Large-Scale Cryo-Electron Tomography of Chlamydomonas Reinhardtii.” Molecular Cell, Elsevier, doi:10.1016/j.molcel.2025.11.029.
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