The internship is part of a larger project which concerns the definition of the 3D structure of proteins forming membrane contact sites, by Cryo Electron Microscopy (EM), in vitro. We are particularly interested in understanding the architecture of the complex formed by VAP-A and OSBP between the endoplasmic reticulum and the golgi, a key complex in the transport of PI4P and cholesterol into the cell. We reconstructed this complex in vitro using purified proteins and model membranes and obtained a model of the two proteins by electron cryo tomography and sub tomogram averaging1.
In order to increase the resolution, we will use a single particle approach (SPA) on the complex in solution. As this complex only forms in the confined space of the contact site, we have designed a DNA origami capable of imposing the proximity of the two proteins and inducing the formation of the complex, which will then be characterized by Cryo-EM.
DNA origami is a technique that uses hundreds of short DNA oligonucleotides, to fold long single-stranded DNA, into various nanoscale architectures. Due to its high degree of spatial customization, DNA origami provides a versatile tool that opens up opportunities for a wide range of applications that require programmed spatial control of molecules in three-dimensional (3D) space. An example is the use of DNA origami for the design of structures used in structural biology for Cryo Electron Microscopy approaches23.
The work proposed for this internship will be done in two parts in two different laboratories:
- the construction of origami from oligonucleotides at the CiTCom laboratory under the direction of Nesrine Aissaoui.
- optimization of protein interaction on origami using biochemistry and electron microscopy approaches, at Institut Curie under the direction of Manuela Dezi
The candidate must have a solid foundation in molecular biology and biochemistry and have a strong interest in structural biology and EM. He/she will be able to benefit from the skills and know-how of the two supervisors as well as from the rich scientific background of the two laboratories and evolve in the field of structural biology. The internship can be followed by a thesis which will aim to obtain the atomic structure of the complex by Cryo-EM and Single Particle Analysis.