Could you remind us briefly what your project is about?
As mentioned earlier in my previous post, I will be studying three membrane proteins; bacteriorhodopsin (BR), Leucine Transporter (LeuT) and outer membrane protein F (OmpF) using neutron macromolecular crystallography (NMX) technique.
One can study the diffraction pattern of a protein in different ways, be it X-rays, electron or neutron. I chose neutron diffraction studies for my proteins. This is because I would like to determine the hydrogen positions and understand the protonated state of different chemical groups in proteins which is otherwise not possible by X-Ray diffraction. But, why it is difficult to locate hydrogen by X-Ray crystallography?
X-Rays interact with matter on the basis of atomic number, thus diffracted intensity is larger for atoms with large atomic number and vice versa. This results in almost negligible diffracted intensity by hydrogen due to low atomic number. On the other hand, neutrons interact with the nuclei, thus no dependency on atomic number. But, this doesn’t solve our problem fully as hydrogen has negative scattering length that usually cause a density cancellation in fourier maps as shown in red colour in Fig 1.
We as scientists find a solution to every problem. Thus, came up with an idea of replacing hydrogen with its isotope deuterium (D2O) either by preparing all buffers in D2O or exchanging during crystal growth. Deuterium coherent scattering length is positive like other common atoms in protein and thus significant noise reduction and good gain in signal. This also reduce the requirement of very big crystals for neutron diffraction as explained by Sam in his previous post. For more insights into neutrons, check a cartoon illustration in Fig 3.
In my project, I will purify and crystallize my protein in deuterated conditions and aim for bigger crystals to finally obtain a neutron structure of a membrane protein. Voila!!
-What important milestone have you reached until now?
So far, I have purified two different membrane proteins, OmpF and LeuT. It took quiet a while to optimise purification of OmpF but thanks to Prof. Poul Nissen’s lab for providing me with an optimized protocol for LeuT production that saved quiet alot of time. I have some beautifully diffracted crystals of OmpF at a resolution of 2A° (Fig 2). But, now the real challenge begins to play with my crystals and grow them bigger by using different crystallization techniques (mostly vapour diffusion). Meanwhile, I did expression trials of OmpF in deuterated minimal media that went well and will be scaled up to get some protein for crystallisation experiments.
– Did the ITN help you in the implementation of your project until now? If yes, in what way?
Yes, definitely. During this 1.5 years of my PhD, I had already worked in two different labs within the network. My first OmpF crystals were diffracted at ESRF while I was on a secondment in Monika Spano’s lab. We also tried getting bigger crystals using dialysis and batch techniques. My second project was initiated in Aarhus, Denmark (at Poul Nissen’s lab) and it was such a learning experience from all aspects amongst so many Postdocs and PhD’s. Switching from few people in my lab to 30 people in Poul’s lab taught me time management and advance planning of my experiments to avoid the competition for instruments (:P)
Would you recommend other students to apply to a position within a MSCA network such as RAMP?
Of course! If you like to move around and love multi-tasking like all of us in RAMP, MSCA is the right platform for you. You develop personally and professionally by attending various workshops and schools. It also helps you to build an international network that will really help you in your scientfic career.
Is there a topic you would like to share/collaborate within the network in relation to your research work?
Since, I am working on a model membrane protein, it’s always a bit easier to play around because you don’t have to worry much about protein stability and yield. So, people who have stability issues and would like to test their experiments are most welcome to use my protein. Apart from that, I would encourage others to go for NMX, if they have some hydrogen related questions. Sam and I are working on methods to grow bigger crystals, hopefully we can standardize the procedure and make others life easier in near future!