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Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes


Dutka, Przemysław (2023) Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/87jm-7v06.


Cryo-electron tomography (cryo-ET) is a powerful method for investigating the 3D structure of intact cells, organelles, and complex protein macromolecules that cannot be crystallized or are too heterogenous for single-particle cryo-electron microscopy (cryo-EM). However, obtaining high- resolution cryo-ET structures for many biologically important targets is still a challenge. To address this challenge, cryo-ET can be combined with other methods, including X-ray crystallography, single-particle cryo-EM, structure predictions, cross-linking mass spectrometry, biochemistry, and evolutionary analysis to produce integrative models. Recently, with the development of AI-based tools such as AlphaFold2, structure prediction has played an increasingly important role in integrative modeling. The combination of cryo-ET and structure prediction in particular has provided unprecedented insights into the ultrastructure of cellular components. This thesis focuses on two bacterial multi-megadalton protein complexes which are difficult to study by classical structural biology approaches: gas vesicles (GVs) and the Legionella pneumophila Dot/Icm type IV secretion system (T4SS). GVs are gas-filled protein nanostructures that regulate the position of certain microorganisms in water and consequently their access to sunlight and nutrients. Here, we investigate the mechanical properties of GVs and reveal the molecular structure of GVs and its implication for the assembly mechanism. The Dot/Icm T4SS is a macromolecular complex formed by approximately 27 proteins, utilized by L. pneumophila to hijack the host cell's biology for its replication purposes. A nearly-complete integrative model of this complex provides crucial insights into its structural organization and its evolution from conjugation to secretion, as well as the transportation of substrates into the host cell.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Gas vesicles, type IV secretion system, cryo-ET, integrative modeling, subtomogram averaging
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Shapiro, Mikhail G. (advisor)
  • Jensen, Grant J. (co-advisor)
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Shapiro, Mikhail G.
  • Jensen, Grant J.
  • Phillips, Robert B.
Defense Date:28 April 2023
Record Number:CaltechTHESIS:05092023-163511031
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter 2 (Measuring Gas Vesicle Dimensions by Electron Microscopy) adapted for Chapter 3 (Geometric Effects in Gas Vesicle Buckling under Ultrasound) adapted for Chapter 4 (Structure of Anabaena Flos-Aquae Gas Vesicles Revealed by Cryo-ET) adapted for Chapter 5 (Structure and Function of the Dot/Icm T4SS)
Dutka, Przemysław0000-0003-3819-1618
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15157
Deposited By: Przemyslaw Dutka
Deposited On:15 May 2023 20:20
Last Modified:22 May 2023 20:33

Thesis Files

[img] PDF - Final Version
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[img] Video (AVI) (GVs' sensitivity to electron dosage) - Supplemental Material
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[img] Video (MPEG) (Building an integrative model of the Legionella pneumonia Dot/Icm T4SS) - Supplemental Material
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