Sam’s blog post

– Could you remind us briefly what your project is about?

As mentioned in my first post, my project is about studying the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) using neutron macromolecular crystallography (NMX). NMX is similar to the X-ray diffraction technique that Diogo explained previously, except we observe how a protein crystal produces a neutron diffraction pattern. As neutrons scatter off atomic nuclei instead of electrons as with X-rays, hydrogens – which have very few electrons for X-rays to scatter off – are more easily revealed.

In the case of SERCA, we are interested in how protons move through the protein during its functional cycle. The protein performs the vital function of controlling intracellular calcium levels by pumping Ca²⁺ from the cytoplasm into to the sarco/endoplasmic reticulum (SER). At the same time, SERCA also counter-transports protons from the SER into the cytoplasm. While we have a good understanding of Ca²⁺ transport, we are unsure of the exact pathway these protons take through the protein. We hope that by mapping hydrogens in SERCA using NMX, we will be able to identify this proton pathway.

However, NMX experiments also present significant challenges, in particular the requirement for large, well diffracting protein crystals. This requirement is due to the very low flux of current neutron sources compared to their X-ray counterparts. Given that diffracted intensity is directly proportional to both the intensity of the incident beam and crystal volume, much larger crystals are needed by NMX to compensate for its weak source. Therefore, I am currently trying to produce well-diffracting SERCA crystals of the size needed to perform these neutron diffraction studies (crystals of about ~1 mm³ in size are needed).

To produce crystals of this size, I am using a variety of crystallisation methods which offer extended crystal growth (a topic for a future post?). So far I’ve managed to increase the size of SERCA crystals to a reasonable extent (Figure 1), and am currently trying to get the extra bit of growth I need to start initial neutron experiments.

– What important milestone have you reached until now?

A significant milestone was achieved when I managed to obtain a room temperature X-ray structure of SERCA using the home X-ray source in our lab (Figure 2).

This was important for several reasons. First, it gave us an understanding of how well the SERCA crystals we produced so far diffracted and how much we need to improve (the crystals diffracted to a resolution of 3 Å and we are aiming for a resolution of 2.5 Å). Second, the process gave me valuable experience in preparing crystals for room temperature diffraction studies (NMX will be performed at room temperature; X-ray studies are normally performed at 100 K, or -173 °C). Finally, we would like to compliment any SERCA structure we get using neutrons with an equivalent X-ray structure; combining the two structures provides much more information that either one alone.  

Another important milestone is that we managed to get our efforts so far towards producing large SERCA crystals published in a scientific journal! Details below:

T. L.-M. Sørensen, S. J. Hjorth-Jensen, E. Oksanen, J. L. Andersen, C. Olesen, J. V. Møller and P. Nissen (2018) Membrane protein crystals for neutron diffraction. Acta Crystallographica Section D Structural Biology, 74: 1208-1218.

– Did the ITN help you in the implementation of your project until now?

Absolutely! Being part of the ITN has allowed me to get advice from experts not just from my home lab here in Denmark, but also from other leading labs and institutes throughout Europe. Recently, I spent 2 months in Lund, Sweden (at Swati’s lab) where I explored the dialysis technique of crystallisation in detail. Being in a different work environment exposes you to new ideas and different perspectives, and you try things you otherwise would not. I found my time in Sweden really helped me figure out what to do next, and I’m very much looking forward to my next secondment at the Université Grenoble Alpes later this year.

– Would you recommend other students to apply to a position within a MSCA network such as RAMP? What advice would you give them?

If you are interested in a career in scientific research, applying for a position within a MSCA network is a great way to start. You get to attend workshops and placements at universities, research institutes, and companies from all over Europe – where you get to learn from the best and brightest in a range of topics within your field of research. You also become part of an international community and develop a professional network that will be invaluable no matter what the next step in your scientific career is.

 – Is there a topic you would like to share/collaborate within the network in relation to your research work?

A significant portion of my project involves growing the very large protein crystals required for neutron diffraction studies. Once we are able to reliably produce well-diffracting SERCA crystals approximately ~1 mm³ in size, it would be great to test the methods and techniques used on the other membrane proteins studied within this network. As mentioned before, NMX has the potential to reveal information (i.e. hydrogens) that no other method is able to and is essential for understanding certain problems regarding protein function. Developing a generalised strategy for producing the large membrane protein crystals needed would help to make NMX more assessable to the wider scientific community, and in turn, help us to better understand how life works at the atomic scale.