Students blog about their activities within RAMP: #research #secondments #outreach #PhDlife
“During the first Irish lock down, I had to transition from working in the lab daily to working from home. I used that time to prepare a literature review, learning about what nucleation is, how it occurs, and the techniques used to study it. Being able to take the time to understand this further allowed me to better understand my project and where it fits in with what we know and don’t know.”
October 2020, Sofia (ESR 5)’s last months before defending: All my attention is focused on writing my thesis
“During my PhD I came across with different disciplines and I met people that inspired me to pursue a career in a high- scientific level. I am not sure if I will pursue an academic or industrial career, but I know that I would like to have a cutting-edge technology Postdoc in physics and engineering applied in membrane protein crystalline samples.”
“As much as I love being in science, sketching is therapeutic for me. So, I would love to combine my hobby and work by being a Science Illustrator in near future. While building my entrepreneurial skills, I will continue being a crystallographer whose always intrigued to learn more about protein structures.”
“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!”
“My project like many others took twists and turns, and was hampered by the recent pandemic that affected all of us worldwide. Now however, I am reaching the final sprint of the project; I have purified and obtained crystals of the mutant version of my current membrane protein target; I have validated that it is in the state that we need it to be; I have troubleshot some additional problems that popped up during the protein production; and am now producing large amounts of the protein for the time-resolved experiments.”
“being in this network has been an important turning point in my life. I’ve gained invaluable educational and life experiences that I know will help me throughout my career. And last, but by no means least, I’d like to say thank you to the European Union for funding this scholarship. It’s had an incredibly positive impact on my life.”
“Like many others, I have had a hard time adjusting to these new working conditions. I have struggled with both motivation and concentration. As such, it has been a conscious and ongoing effort to find out what works best for me – learning what times of day I’m most productive, how to block out distractions, and how to break work up into small, achievable pieces. The technology that we have today has made the situation easier than it otherwise would be.”
“We all hope that we will be able to resume our everyday life, meet family and friends as soon as possible. However, we should not forget a few lessons from the Covid-19 pandemic and maybe one of them is how important science communication is.
I reckon institutions should give more support and resources to scientists to help them invest their time in it and science should have a more active role in policy making. This would allow scientists not only to be able to share their research more effectively to the general public, but also to contribute to informed decision-making worldwide.”
“By late February, everything was ready for the conference: the schedule, the website, speakers and students accommodation, the catering. It was to be held in University of Southampton, on the 25th to the 27th of March. At the same time, more and more news started to emerge about what had started as a concentrated epidemic in a region in China, now spreading rapidly to the rest of the word. One after the other, big conferences and scientific meetings began announcing their cancellation. By early-March, we were still maintaining some hope, that since we were going to be a small conference we will be able to go ahead. Unfortunately, as we were getting closer to the dates, we realised that we should face the situation sensibly and cancel the conference until further notice.”
January 2020, Sofia (ESR 2): Crystallisation is essential for RAMP and she has a unique way of doing it: microfluidics
“The microchips that I am developing use materials that are compatible for in situ X-ray measurements. This means that we don’t have to harvest the protein crystals and the crystals won’t be damaged during the steps of harvesting or cryocooling. So basically, we can use one single device in order to crystallize membrane proteins and directly use them for on-chip and in situ X-ray diffraction experiments. Another advantage of microfluidic crystallization is to significantly reduce the amounts of protein samples to a few hundred nanoliters.”
“Actually, my project changed quite a bit from what I was doing in the beginning. I started a really ambitious project, aiming to not only crystallise a membrane protein on its own but bound to some important cellular binding partners. This sounded amazing, but also included a lot of challenges, ranging from getting the individual protein components, to isolating the complex and finally crystallise this thing. I was already struggling with the first step and soon had to admit that the goals set in the beginning would probably not be met. However, I made some good progress with some side projects I started during my secondments in Hamburg and at Novartis, which I could combine to create a new story for my thesis.”
“In Hamburg, I got much more experienced in collecting data at synchrotrons. Additionally, I learned how to process the data to get a structure and refine it. At Molecular Dimensions, I had to change my perspective and put myself in the supplier’s shoes. I was involved in, and learned about, the design, pricing and production of a product, which was very exciting and new to me.”
“Being involved in other projects,
Maintaining and establishing new collaborations,
“All the talks were absolutely brilliant. Some speakers used props and demos to explain the key concepts, that made the presentation very catchy and interactive and allowed us to engage the audience as much as possible. Even though the talks were prepared separately and we only had a couple of evenings for “rehearsal” before the event, the story flowed perfectly from one topic to the following one.”
“The main part of my PhD involves building computational models, but it is the first time that I am applying my coding skills to build a web tool. Since I started my secondment, I have learnt a lot of new skills and evolve my coding knowledge by working in a completely different project.
Another interesting aspect is the collaboration with a company. We are designing a product that will eventually be used by a wide audience. I have to build a tool that will satisfy the needs of the client, but at the same time, it will be easy to use and can be implemented in a limited amount of time.”
“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.”
“The purpose of my secondment at the Institute for Structural Biology in Grenoble, France is to try to grow large crystals of SERCA for NMX studies using some of the specialised equipment they have here, in particular an apparatus which allows the temperature of the crystallisation mixture to be finely controlled.
The idea is to alter the protein solubility of the mixture by changing the temperature – this means that you can adjust the conditions to the point where the protein contained within the solution will be added to the mass of the existing crystal (or crystals) instead of coming out of the solution as new crystals. The result is a few very large crystals instead of many smaller crystals.”
“During the week at Imperial College in London, we were trained on writing publications (papers/reports/thesis) and understanding the process of manuscript review. We spent time on discussing the importance of a career planning, the preparation of effective CV’s, the application procedure and interviews. Finally, we were trained on public engagement and the significance of communicating research in general public and schools. During the week at the University of Surrey, we had the opportunity to find out what kind of leaders we are or we can be, how we can lead within teams and how we can follow other leaders and influence the general public.
Overall, this workshop provided a lot of tips that I believe that sooner or later will be useful for all of us, no matter the career choice that we will make.”
“I really would like to get the chance to go back again. I will see how it goes to reproduce the crystals I got there here back in Leeds. If this shows is too difficult it would be great to go back to the already evaluated setup.”Claudia
“Even though the project of my secondment has been completed. There are several reasons that I would really like to return. First of all to meet and work again with the people that I collaborated. Secondly to expand this project and try some Cryo EM experiments on the GPCR target in complex with various components and other proteins. Furthermore, If I had the opportunity, I would like to try some lipidic cubic phase crystallization set up on these targets.”Sofia
“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.
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!!”
“Protein crystals are softest in the group. Compared to water molecules (3 atoms), proteins are absolutely huge molecules (1000s atoms), which in crystal form interact with one another with a diverse array of weak interactions. In addition, proteins are lumpy and so they don’t pack together very well. The gaps between individual protein molecules are filled with liquid. This means they are more like a cube of jello than a diamond or ice crystal.”
April 2019, Jannik (ESR 4): Last news on solving mPPases structures and Jannik’s view on the MSCA network
“I can definitely recommend joining a MSCA network! RAMP opened many doors for me and gave me the opportunity to kick off my scientific carrier with a big network of excellent researchers across Europe. The secondments help to extend your network even further and allow you to complement your set of skills. If you want to do a PhD, I recommend to definitely start by looking for MSCA networks that fit your interests first. I am a big fan!”
Mach 2019, My (ESR 9)’s first secondment at Imperial College London in the lab of Prof. Bernadette Byrne
“I had the chance to get an insight into a lab with experience in membrane protein crystallization and learned about expression, purification and stability assays for α-helical membrane proteins using the mosquito® crystallization robot for commercial crystallization screening approaches, the Rigaku X-ray generator and other crystallization facilities.
These were hands-on experience, which you can’t acquire by doing literature research and the value of it is essential for my PhD. Based on this experience I have built up confidence in applying the methods for a new model membrane protein.”
“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!”
“For this kind of project, one of the bottlenecks is the amount of pure protein required, as well as its successful transformation into protein crystals. Because of this, a large amount of my work is not the final time-resolved experiments, but also all the preceding effort and labor involving purification and crystallization of proteins – any advance in this sector will have a profound effect on the final desired experiments.”
“As a physicist it was a challenge to work on a biochemistry lab and keep up with the everyday challenges of cell growth and protein production. My first goal was to get familiarized and expertise all these techniques. This was the first important milestone that I succeeded. Another challenge was the creation of the phase diagram.
Large amount of protein have been used and being economical with protein consumption such as finding minimal, reproducible volumes from the pipettes was an obstacle that needed to be overcome. Finally, I completed a diagram and now the next step will be to apply it to more membrane proteins and test its reproducibility for them.”
“I had the chance to be in charge of managing every single step of the product development, from the theory to practice, from deciding the different materials and costing them to arranging product development meetings with collaborators from different countries (and continents!) and planning a “field trip” to perform the procedure.
Networking, working with collaborators, project management and problem solving are all essential skills for a researcher, so the more opportunities we have to practice them, the better!”
“Instead of doing an experimental study on how membrane proteins crystallise, I will try to create computational models to understand the physics of the crystallisation of such complex biological systems. That means studying nucleation and the growth of crystals, in correlation with phenomena such as diffusion and flow, that play an important role in the crystallisation process.
The experimental ESRs will help me to achieve my goal, by providing experimental data that they collected during their experiments. Then by analysing their data, I will be able to find patterns and trends that will help to better understand how membrane proteins crystallise.”
“MSCA networks provide more than scientific opportunities. I am learning how to better communicate with people of various backgrounds, how to make new acquaintances and build my network. I learned to better deal with deadlines and administrative procedures. And last but not least, during my secondments within the RAMP network, I learned how to adapt to new conditions faster and more efficiently.”
“I now am able to produce several versions of my protein which I can purify and use to try and form the complex with.
In a parallel approach, I am directly producing both proteins of the complex inside of the same cell. By attaching fluorescent signals to the proteins, I was able to visualise first evidence of complex formation.”
“As an ESR you should actively engage in the workshop and ask lots of questions. I think we did a good job and since we were often split into a set of smaller groups, there was always enough time for everyone to ask questions if something was not clear.”
“Methods in macromolecular crystallization”. This course was about different methods in crystallization and in crystallography studies. It was completely related to my thesis and gave me a general understanding of various methods in crystallization of the protein and in methods for studying their structure and troubleshooting.
“This workshop was all about how we crystallize membrane proteins (MP) in presence of lipids and make the in vitro niche most suitable for the crystallization of a protein. In order to achieve this, we gained the expertise in Hi-LiDe and Lipidic cubic phase (LCP) crystallization methods at Trinity College, Dublin. We also learnt the importance of protein solubility and protein-protein interaction in crystallization and how we can measure it by a technique called dynamic light scattering at University of Maynooth.”
June 2018, My (ESR 9)’s experience in the recent RAMP research workshop “in meso in depth” at Dublin & Maynooth
“I enjoyed learning new expertise, which were not directly related to my project now but may be helpful in the future. Having the opportunity to see the remote access to the synchrotron at SLS for example was very impressive.”