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Structure and Function of the Dot/Icm T4SS

Dutka, Przemysław and Liu, Yuxi and Maggi, Stefano and Ghosal, Debnath and Wang, Jue and Carter, Stephen D. and Zhao, Wei and Vijayrajratnam, Sukhithasri and Vogel, Joseph P. and Jensen, Grant J. (2023) Structure and Function of the Dot/Icm T4SS. . (Unpublished)

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The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Dutka, Przemysław0000-0003-3819-1618
Liu, Yuxi0000-0003-1439-9000
Maggi, Stefano0000-0003-4853-5840
Ghosal, Debnath0000-0002-2227-0330
Carter, Stephen D.0000-0002-4237-4276
Zhao, Wei0000-0002-4441-9722
Vijayrajratnam, Sukhithasri0000-0001-7545-6510
Vogel, Joseph P.0000-0002-8054-5021
Jensen, Grant J.0000-0003-1556-4864
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech and Pacific Northwest Center for Cryo-EM (PNCC). We thank the Liu lab for sharing data and expertise in using I3 to resolve the DotO hexamer of dimers. Y.L. was supported by a Jane Coffin Childs Memorial Fund for Medical Research post-doctoral fellowship. This work was supported by the National Institutes of Health (grant R01-AI127401 to G.J.J.). AUTHOR CONTRIBUTIONS. P.D. prepared samples, reconstructed tomograms and picked particles for a subset of data, performed subtomogram averaging, performed data exploration, located proteins forming the system, participated in building the model, drafted the manuscript, and prepared the figures. Y.L. conceived experiments, prepared samples, acquired and reconstructed tomograms, picked particles and worked on subtomogram averaging, performed the particle shift analysis, planned and led FIB-milling and infection experiments, and contributed to writing of the manuscript. S.M. performed data exploration, identified proteins forming the system, led model building, prepared the movie explaining the model building and helped write the manuscript. D.G. conceived experiments, prepared samples, acquired and reconstructed tomograms, and helped interpret the data. J.W. reconstructed tomograms, picked particles for a subset of data, worked on FIB-milling and infection experiments, performed segmentation, participated in data interpretation, and helped to write the manuscript. S.D.C. helped plan and execute the infection experiments. W.Z. helped plan and execute the FIB-milling experiments. S.V. constructed L. pneumophila strain JV9298. J.P.V. constructed L. pneumophila strain JV9082 and helped revise the final version of the manuscript. G.J.J. supervised the entire project from experimental design through interpreting the results and helped write the manuscript. DATA AVAILABILITY. Cryo-ET maps and Dot/Icm T4SS integrative models will be uploaded to public repositories and made available upon publication. During revision process data are available upon request. Scripts used for data analysis are available on Zenodo:,, The authors have declared no competing interest.
Funding AgencyGrant Number
Jane Coffin Childs Memorial Fund for Medical ResearchUNSPECIFIED
NIHR01 AI127401
Record Number:CaltechAUTHORS:20230324-517798000.1
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:120378
Deposited By: George Porter
Deposited On:29 Mar 2023 03:41
Last Modified:29 Mar 2023 03:41

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