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High resolution cryo-electron microscopy of clathrin cage networks


School of Life Sciences

About the Project

This project is available through the MIBTP programme on a competition basis. The successful applicant will join the MIBTP cohort and will take part in all of the training offered by the programme. For further details please visit the MIBTP website - https://warwick.ac.uk/fac/cross_fac/mibtp/

In cryo-electron microscopy, the superior signal sensitivity of new direct electron detectors has revolutionised the field of structure determination allowing sub-4Å structures of challenging targets such as membrane proteins and ribosomes to be obtained without using X-ray crystallography. We are exploiting this improvement in capability to carry out high resolution structural analysis of clathrin cage complexes (Morris et al., 2019)

Our aim is to understand how the proteins involved in the network of clathrin and its adaptor proteins interact to achieve clathrin-coated vesicle formation. Clathrin-mediated endocytosis is a fascinating mechanical phenomenon that drives the selective internalisation of molecules into cells. Nutrient uptake, synaptic vesicle recycling, signalling, determination of cell polarity and development all rely on endocytic mechanisms. In disease, viral and bacterial pathogens exploit endocytosis to gain entry into cells and malfunctions lead to tumour formation, neurodegeneration and heart disease. In order to work properly, clathrin-mediated endocytosis requires accurate and timely assembly of a clathrin lattice and coordination with a network of more than 20 adaptor proteins to form a coated vesicle which will be able to select molecules from the outside of the cell for delivery to specific destinations.

In this project you will use high resolution 3D cryo-electron microscopy to visualise adaptor proteins binding to clathrin cages and biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy, surface plasmon resonance (SPR, Biacore) and isothermal titrating calorimetry to investigate how clathrin-adaptor interactions result in formation of a functional coated vesicle network. You will have access to the Gatan K2 Summit direct detector and Jeol 2200FS 200kV transmission electron microscope provided by the Advanced Bioimaging Research Technology Platform, the Titan Krios microscope available in Leicester through the Midlands Regional Cryo-EM Facility and excellent facilities for biophysical analysis available within Warwick School of Life Sciences. This is a fabulous opportunity to apply cutting edge techniques to discovering how clathrin and its adaptor proteins drive clathrin mediated endocytosis.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Plant Science & Structural Biology. Sustainable Agriculture and Food: Plant and Crop Science.

Techniques that will be undertaken during the project:
 High resolution electron microscopy
 Image analysis of large data sets
 3D imaging of structural data
 Kinetic analysis using light scattering, fluorescence and single molecule methods.
 Protein mutagenesis, expression and purification

References

Morris KL, Jones JR, Halebian H, Wu S, Baker M, Armache J, Ibarra AA, Sessions RB, Cameron AD, Cheng Y, Smith CJ. Cryo-EM of multiple cage architectures reveals a universal mode of clathrin self assembly. Nature Structural and Molecular Biology (2019) In press.


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