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Cryo-EM Analysis of TcdB in Complex with Host Cell Receptors


EMSL Project ID
50994

Abstract

Clostridioides difficile infection (CDI) is responsible for the majority of hospital acquired diarrhea cases reported in the United States. The pathology of CDI relies on the activity of three different toxins, toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT), with TcdB being the most potent. We propose to investigate the structure of TcdB in complex with host cell receptors with a specific interest in understanding how TcdB utilizes the Frizzled (Fzd) family of proteins to mediate toxicity. To this end we have purified a construct of TcdB amenable for cryo-EM analysis and have found vitrification conditions under which we observe a large number of high contrast views of these particles. From these data we have obtained high resolution reconstructions of TcdB in the apo and Fzd bound forms. Through this work have determined an optimal processing strategy which uses a MultiBody refinement approach to obtain reconstructions of heterogeneous portions of TcdB. We now propose probing the structure of TcdB in complex with the predicted Fzd co-receptor, Wnt. Understanding these interactions will lead to valuable insights into the function of TcdB in the context of infection. For these studies we are requesting access to a Titan Krios equipped with an energy filter and phase plate in order to collect a large dataset that we will independently process.

Project Details

Start Date
2019-08-01
End Date
2020-07-14
Status
Closed

Team

Principal Investigator

Dana Lacy
Institution
Vanderbilt University Medical Center

Team Members

Jue Wang
Institution
Vanderbilt University

Jaime Jensen
Institution
Vanderbilt University Medical Center

Michael Sheedlo
Institution
Vanderbilt University

Harry Scott
Institution
Oregon Health & Science University