Sam’s update on his Serca crystals

Could you remind us of what your project is about?

As previously mentioned, my main project is about growing very large crystals of SERCA so that they can be analysed using a technique called neutron diffraction. However, recently I have been focused on a couple of side projects. One project involves using X-ray crystallography to investigate the specific interactions between novel inhibitors and SERCA. These inhibitory compounds are photoactive (i.e. they change their properties upon stimulation with light) and the idea is to be able to switch SERCA activity on and off by applying light.

Another project requires producing microcrystals of SERCA in order to perform a time-resolved X-ray crystallographic experiment. As SERCA transports calcium using the energy obtained from ATP hydrolysis, the idea is to crystallise SERCA with a modified version of ATP that can only be broken down once it is activated with a UV laser. By varying the time between the laser and the X-ray beams, it is hoped to be able to obtain structures of SERCA at different stages of the reaction. This information will help us to understand the activity of SERCA at a more detailed level.

Could you give us a brief update on your project?

My project was also disrupted by the coronavirus pandemic which has affected us all. I was just finishing my two-month secondment at AstraZeneca in Sweden when the widespread lockdown started. Fortunately, I was able to make to back to Denmark safely but everything had closed by the time I had arrived. Not being able to go into the lab meant I was unable to perform the experiments needed to progress. Though I quickly got used to working from home during this period (and even enjoyed it to some extent), there was only so much to be done from the computer. Luckily, the situation here in Denmark had improved to the point where we were allowed full access to the lab again from late May.

Once I had returned to the lab, I focused on testing assessing the inhibitory properties of the photoactive compounds under different wavelengths of light. The results seem to suggest that there is a small difference in SERCA activity depending on what light wavelength is used. Luckily, I had manged to get X-ray structures of SERCA with two of the compounds so now I am trying to explain the activity results using this structural information.

Growing microcrystals of SERCA. (A) The crystallisation experiment involves mixing protein and precipitant solutions together in a microtube, then incubating the mixture at controlled temperature. (B) An image of the resulting crystals.

Now it is June and I am focused on the time-resolved X-ray crystallography experiment. Just before the lockdown I managed to produce microcrystals of SERCA with the modified ATP and sent them off to Hamburg to be analysed. Unfortunately, the facility had shut down by the time my crystals had arrived, and by the time they had reopened, my crystals had not survived the extended time in storage. I have now replicated those microcrystals and plan to test them in a few days. I hope that they will diffract well!

Finally, I am finalising my main project of growing very large crystals. It is going to be stressful trying to finish all these projects in the lab over the next couple of months. I would like to think that every experiment will work out perfectly moving forward but we all know that’s not going to happen 🙂 But that’s what being a PhD student is all about – overcoming whatever challenge stands in your way!