One of the grand challenges of structural biology is to produce atomic level understanding of a biological cell. Towards this goal, we would like to decipher atomic structures of all macromolecules in a prokaryotic cell using electron cryotomography (cryo-ET) and subtomogram averaging, combined with mass spectrometry performed in a novel instrument with bespoke hardware.
We are looking for an ambitious student who would like to learn and apply cryo-ET to produce atomic structures from prokaryotic cells de novo. While cryo-ET can provide pictures of specimens at high spatial resolution, different macromolecules cannot be easily distinguished from each other in black and white microscopy images. To circumvent this problem; mass spectrometry (MS) is a structural biology technique that allows the exact chemical identity of molecules in a sample to be determined. The second part of this project to study the molecular landscape of a cell will combine EM and MS imaging to accurately identify molecules inside cells. Combining high-resolution structures solved directly from cells with spatial MS data will be used to decipher a high-resolution snapshot of a cell.
This proposed project represents basic research, using the latest hardware and software advances in cryo-ET and MS. It is expected that once the workflows are established, they will be applied to important biological specimens for obtaining unique physiological insights into phenomena at the cellular level. This project is ideal for a student interested in microscopy, with an indomitable spirit of exploring uncharted waters. More details will be provided during the interviews.
There is flexibility in biological direction of the project depending on the interests and expertise of the ideal student. Broadly, we are studying the cell surface and cellular organisation of bacteria and archaea important in shaping biofilms and microbiomes. There will be opportunities within the project to learn focused ion beam milling of biological specimens, electron tomography (cryo-ET) and advanced data analysis techniques including subtomogram averaging structure determination from cells.
Our laboratory is friendly and highly collaborative, with details of the research projects almost always arising from brainstorming sessions and lively discussion between lab members and other members of the LMB. There is ample support in our lab and at the LMB for microscopy, image processing, MS and bioinformatics directly available to the candidate. We welcome applications from students of all scientific backgrounds.
References
J Böhning, TAM Bharat, SM Collins. (2022)
Compressed sensing for electron cryotomography and high-resolution subtomogram averaging of biological specimens.
Structure 30 (3), 408-417. e4
Jasenko Zivanov, Joaquín Otón, Zunlong Ke, Kun Qu, Dustin Morado, Daniel Castaño-Díez, Andriko von Kügelgen, Tanmay AM Bharat, John AG Briggs, Sjors HW Scheres. (2022)
A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0.
BioRxiv.
J Böhning, TAM Bharat (2021)
Towards high-throughput in situ structural biology using electron cryotomography.
Progress in Biophysics and Molecular Biology 160, 97-103.
Andriko von Kügelgen, Haiping Tang, Gail G Hardy, Danguole Kureisaite-Ciziene, Yves V Brun, Phillip J Stansfeld, Carol V Robinson, Tanmay AM Bharat. (2002)
In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.
Cell 180 (2), 348-358. e15.
TAM Bharat, SHW Scheres (2016)
Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION.
Nature protocols 11 (11), 2054-2065.
Key papers on using EM and MS for biology -
Tim K Esser, Jan Böhning, Paul Fremdling, Mark T Agasid, Adam Costin, Kyle Fort, Albert Konijnenberg, Alan Bahm, Alexander Makarov, Carol V Robinson, Justin LP Benesch, Lindsay Baker, Tanmay AM Bharat, Joseph Gault, Stephan Rauschenbach (2022)
Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition.
PNAS Nexus (in press).
Melia, C., Bolla, J.R., Lanwermeyer, S.K., Mihaylov, D., Hoffmann, P.C., Huo, J., Wozny, M.R., Elfari, L.M., Böhning, J., Owens, R.J., Robinson, C.V., O’Toole, G.A., Bharat, T.A.M. (2021)
Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition.
Proceedings of the National Academy of Sciences 118 (31), e2109940118.
Chorev, D.S., Tang, H., Rouse, S.L., Bolla, J.R., von Kügelgen, A., Baker, L.A., Wu, D. Gault, J., Bharat, T.A.M., Matthews, S., Robinson, C.V. (2020)
The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes.
Nature protocols 15 (5), 1690-1706.