Can you remind us briefly what your project is about?
The topic of my PhD thesis is “Optimisation of crystal growth using a microfluidic technology-based crystallisation bench”
Three main objectives for this PhD are 1. Adapting crystallization bench (OptiCrys) to crystallize membrane proteins 2. Doing a statistical study to compare diffraction resolution of crystals of model membrane proteins, which are obtained using different crystallization techniques 3. Growing large enough crystals for Neutron Macromolecular Crystallography.
OptiCrys (shown in figure 1) was designed in our lab several years ago for crystallizing soluble proteins. This machine uses dialysis method (figure 1b) to crystallize proteins by changing temperature and reservoir composition during the experiment in order to optimize crystallization conditions. It will help us explore different conditions with the same protein sample since the sample is not consumed during the experiment. By using the dialysis method and controlling the temperature and composition of the crystallization solution, we are able to control the crystallization process to have crystals with the appropriate size and good diffraction qualities for X-ray or Neutron Crystallography.
Figure1. a. Crystallization Bench. b. Temperature-controlled flowing reservoir dialysis setup .
My PhD focuses on optimizing the crystallization conditions of two model membrane proteins (ShuA and AcrB). In addition to the crystallization bench, I also use hanging drop vapour diffusion(figure 2) and microdialysis (figure 3) methods. Each technique achieves a different kinetic pathway in the phase diagram and, therefore, the morphology and quality of formed crystals can be different. I will compare the diffraction resolution of the large number of crystals obtained in order to analyse the efficiency and the advantages of each tested technique in order to choose which method is better for crystalizing these model protein targets and how we can improve their crystal quality and crystal size for X-ray and Neutron Macromolecular Crystallography.
Figure 2. Hanging drop method. The droplet, which is placed on a siliconized glass, contains protein and precipitant solution .
Figure 3. Microdialysis method. The droplet only contains a protein sample. The button is covered with a dialysis membrane and placed inside a precipitant solution .
What important milestone have you reached until now?
For this kind of study, we need to acquire pure protein. I work with two model membrane proteins, and now I can successfully purify them. It took a long time, as each protein has its own characteristics and needs different conditions to stay in its native state.
As already mentioned, two proteins I am working with are AcrB and ShuA. AcrB is located in the inner membrane of gram-negative bacteria like Escherichia coli. It exports antibiotics, drugs, and toxic compounds out of the cell. On the other hand, ShuA is located on the outer membrane of Shigella dysenteriae and is used for the transport across the membrane. This protein is a typical β-barrel and contains 22 antiparallel β-strands.
We are still improving the method to increase protein yield, but we have enough biological material for our study now.
We have succeeded in producing, purifying and obtaining crystals with both proteins, even though the quality of diffraction still needs to be improved in the case of AcrB.
Important milestone that we reached recently is having crystals of ShuA that diffract around 2.8 Å resolution. The next step for ShuA will be increasing the size of crystals, while maintaining and/or improving the diffraction quality, to use them for neutron crystallography.
Did the ITN help you in the implementation of your project until now?
In the RAMP network, we have several secondments during our PhD. I’ve visited different labs and learned different techniques related to my work. I believe being part of this network is really helpful, as I’ve had the opportunity to work in different labs and learn from other researchers who work in the same area of study, but with different points of view.
– Would you recommend other students to apply to a position within a MSCA network such as RAMP? What advice would you give them?
I recommend these positions to those who really care about science and like to work in a network. It is a great opportunity to work as a member of this team because not only you are actively researching, but by participating in different workshops and training, you are also preparing for your future career in research.
 Junius, N., Oksanen, E., Terrien, M., Berzin, C., Ferrer, J.L. and Budayova-Spano, M., 2016. A crystallization apparatus for temperature-controlled flow-cell dialysis with real-time visualization. Journal of applied crystallography, 49(3), pp.806-813.
 Russo Krauss, I., Merlino, A., Vergara, A. and Sica, F., 2013. An overview of biological macromolecule crystallization. International journal of molecular sciences, 14(6), pp.11643-11691.