Structure and dynamics of bacterial chemotaxis signalling array by cryoEM

Project Overview

Chemosensory core complex, conformational states and array. (A) The core complex is composed of 2 ...

Bacterial chemotaxis response is crucial for colonization and infection, and the signal transduction systems that mediate such responses are potential new targets for antimicrobial drug development. Such system has emerged as a paradigm for understanding the principles of intracellular signal transduction both in bacterial and eukaryotic cells. In bacterial cells, hundreds of basic core signalling units consisting of three essential components, the chemoreceptors, the histidine kinase and the adaptor protein, assemble into a two-dimensional lattice array which allows cells to amplify and integrate many varied and possibly conflicting signals to locate optimal growing conditions. We aim to determine the structure and dynamics of the chemotaxis signalling arrays using state-of-the-art cryo-electron microscopy and tomography. We will take both in vitro and in situ structural approaches and combined with large-scale all atom molecular dynamic simulations. The ultimate goal is to assemble a time-resolved molecular movie of the entire signalling pathway in bacterial chemotaxis at an atomic level.

Training Opportunities

We are located in the Division of Structural Biology, Wellcome Trust Centre for Human Genetics, which provides an ideal environment for multidisciplinary and integrative studies. We also have regular access to eBIC and Diamond Light Source for data collection. Individual projects are tailored to particular student interests and cover techniques in molecular, cellular and structural biology. Through the projects, students will be trained in

Theme

Protein Science & Structural Biology

Admissions

Project reference number: 927

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Peijun Zhang Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR peijun@strubi.ox.ac.uk
Professor Mark Sansom Department of Biochemistry University of Oxford GBR

Cassidy CK, Himes BA, Luthey-Schulten Z, Zhang P. 2018. CryoEM-based hybrid modeling approaches for structure determination. Curr. Opin. Microbiol., 43 pp. 14-23. Read abstract | Read more

Recent advances in cryo-electron microscopy (cryoEM) have dramatically improved the resolutions at which vitrified biological specimens can be studied, revealing new structural and mechanistic insights over a broad range of spatial scales. Bolstered by these advances, much effort has been directed toward the development of hybrid modeling methodologies for the construction and refinement of high-fidelity atomistic models from cryoEM data. In this brief review, we will survey the key elements of cryoEM-based hybrid modeling, providing an overview of available computational tools and strategies as well as several recent applications. Hide abstract

Cassidy CK, Himes BA, Alvarez FJ, Ma J, Zhao G, Perilla JR, Schulten K, Zhang P. 2015. CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling. Elife, 4 Read abstract | Read more

Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of the molecular events underlying signaling, however, has been hampered by the lack of a high-resolution structural description of the extended array. Here, we report a novel reconstitution of the array, involving the receptor signaling domain, histidine kinase CheA, and adaptor protein CheW, as well as a density map of the core-signaling unit at 11.3 Å resolution, obtained by cryo-electron tomography and sub-tomogram averaging. Extracting key structural constraints from our density map, we computationally construct and refine an atomic model of the core array structure, exposing novel interfaces between the component proteins. Using all-atom molecular dynamics simulations, we further reveal a distinctive conformational change in CheA. Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function. Hide abstract

Fu X, Himes BA, Ke D, Rice WJ, Ning J, Zhang P. 2014. Controlled bacterial lysis for electron tomography of native cell membranes. Structure, 22 (12), pp. 1875-82. Read abstract | Read more

Cryo-electron tomography (cryoET) has become a powerful tool for direct visualization of 3D structures of native biological specimens at molecular resolution, but its application is limited to thin specimens (<300 nm). Recently, vitreous sectioning and cryoFIB milling technologies were developed to physically reduce the specimen thickness; however, cryoET analysis of membrane protein complexes within native cell membranes remains a great challenge. Here, we use phage ΦX174 lysis gene E to rapidly produce native, intact, bacterial cell membranes for high resolution cryoET. We characterized E gene-induced cell lysis using FIB/SEM and cryoEM and showed that the bacteria cytoplasm was largely depleted through spot lesion, producing ghosts with the cell membranes intact. We further demonstrated the utility of E-gene-induced lysis for cryoET using the bacterial chemotaxis receptor signaling complex array. The described method should have a broad application for structural and functional studies of native, intact cell membranes and membrane protein complexes. Hide abstract