In line with the advanced technical training that is a core feature of the Doctoral training program, we are exploring way to advance each of the 3 key areas in the workflow for cryo-EM of GPCRs from enabling biochemistry, to efficiency and robustness of vitrification and imaging, through to optimised data processing and the ability to directly derive 3D conformational dynamics to provide new opportunities for drug discovery initiatives.
This Program Area is available at all Nodes.
Grid preparation and vitrification (All Nodes)
This project area will investigate the impact of different vitrification methods, different grid coatings, and combinations thereof, on the vitrification of exemplar membrane proteins solubilised in different media (detergents, nanodiscs etc), including assessment on partitioning of the particles across the air-water interface, particle stability, orientation distribution of particles, and resolution of 3D reconstruction. The goal of this work is the identification of novel combinations of grid coatings (with distinct wetting properties) and solubilisation methods that can provide enhanced robustness and resolution of membrane protein samples.
Projects in this area are offered by all CCeMMP Nodes, however, some specialised projects may also be offered in a Node-specific manner including (i) collaboration with researchers experienced in surface chemistry and the specific immobilization of biomolecular systems on micro- and nano-fabricated structures; this project will develop approaches to specifically functionalise microscope grid substrates to precisely control the density and orientation of macromolecular complexes (Univ. Wollongong), and (ii) collaboration with the Monash Nanofabrication Centre to generate unique grid coatings (Monash Univ.).
Cryo-EM imaging, data processing, and 3D-reconstruction (All Nodes)
Cryo-EM is in a constant state of evolution. This extends from updates to microscope hardware, detectors, software to support data collection workflows and the processing of cryo-EM data. Within the CCeMMP we have projects that address the integration of the latest software to collection and analysis of membrane protein cryo-EM data. We work with software developers to address the particular complexities associated with small membrane proteins, and the extraction of embedded conformational dynamics from our data. One area of interest is the use of AI to accelerate cryo-EM workflows* (Monash Univ.).
*Applicants interested in this project area will be required to have an appropriate background in mathematics or physics.
Enhancing biochemical approaches to expand the repertoire of GPCR states that can be studied by cryo-EM
While robust methods have been developed for complexes of GPCRs and select G proteins (particularly Gs and Gi/o), optimisation of tools and/or conditions for routine complex formation with other G proteins and other transducers (eg. Arrestins) is required to fully exploit the potential of cryo-EM to understand agonist binding and receptor activation. Beyond this, there is a need to develop or optimise methods to adopt cryo-EM for study of other receptor states, including inactive/inhibitor bound receptors, apo receptors, receptor dimers and complexes of GPCRs with non-canonical G proteins (or other transducers) for structural interrogation of biased agonism.
Solubilisation and membrane mimetic environments
To date, almost all GPCR cryo-EM structures have been solved in detergent solubilised micelles. This project area will explore reconstitution into lipidic environments (eg. Using nanodiscs, SMALPs) and the influence of different lipid composition on formation and stability of GPCR complexes and the conformational dynamics of these complexes.
The instability of unliganded GPCRs has made determination of apo GPCR structure refractory to current approaches. Cryo-EM has the potential to address this gap in understanding of GPCR structure-function through innovation in sample preparation, imaging methods and data processing.
While cryo-EM has become the method of choice for active-state, transducer-bound, GPCR complexes, inhibitor-bound, inactive structures have been the domain of x-ray crystallography. New tools and recent technical developments in imaging and analysis mean that cryo-EM is set to revolutionise the approach to inhibitor-bound structures.
Higher order oligomers and application to drug discovery
With the exception of recent structures of obligate dimers of class C GPCRs, the structural work to date has been limited to monomeric GPCR complexes. Structures of GPCR homo and hetero dimers could open up new avenues for novel drug development.
Structural basis for biased agonism
Biased agonism describes the ability of different ligands acting at the same receptor to differentially promote the recruitment of transducer proteins. To date, success in generation of stable complexes for structural imaging has been almost exclusively limited to the best coupled (canonical) G protein partner. However, to properly understand why one drug can favour a distinct pattern of transducer recruitment relative to another, structure of ligand-receptor complexes bound to different transducers is required. This poses questions on how to generate stable complexes for less well coupled G proteins/arrestins. Success in this project area has the potential to enable design of new drugs with predictable patterns of biased agonism.
Integrating cryo-EM and single-molecule spectroscopy
The van Oijen research group in Wollongong is a world-leading team in the area of single-molecule fluorescence imaging and spectroscopy. We aim to develop methods to visualise conformational motions of membrane channels and receptors in real time using single-molecule fluorescence resonance energy transfer methods and relate those conformational dynamics to high resolution structural information using electron-dense labels obtained through cryo-EM. We will develop approaches that allow structural characterisation of unstructured protein regions hitherto inaccessible via cryo-EM through integration with high time-resolution fluorescence spectroscopy.
Enhancing biochemical approaches to expand the repertoire of signalling receptors that can be studied by cryo-EM
This project area aims to develop robust membrane protein biochemistry purification methods to purify signalling receptors and signalling receptor complexes. In addition, tools (nanobodies/small molecule modulators) will be developed and optimised to capture receptor conformational states. The overall goal of this work is to develop and optimise cryo-EM methods to study receptor conformational states to interrogate signal transduction function, including inactive bound receptors, ligand/inhibitor/ bound receptors, apo receptors, receptor dimers, receptor oligomers.
The overarching goal of this key area will be to explore the capability of cryo-EM to generate 3D-structures of novel Receptor:Drug complexes to support drug discovery and development projects.
Solubilisation methods for cryo-EM
This project area aims to develop robust membrane protein biochemistry purification methods to purify and solubilise membrane proteins, including detergents, nanodiscs, peptidiscs, Saposin-lipid nanoparticles, SMALPS, Amphipols.