Diogo Melo’s blog post

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

As I explained in my first post my project is about obtaining many successive 3-D protein pictures over a wide rande of time-scales (down to every 0.000 000 000 000 001 seconds – femtoseconds). We can then pool the pictures together following the timeline, in the end obtaining a movie of the protein in action. Bird figure.

Figure 1f

At the time I gave an analogy of taking pictures of a bird versus a protein, in the end having a movie of the bird flying, or the protein performing its function, but bear in mind the different size scales (Fig. 1 and Fig 1f): the protein I work with is only 90 angstroms in its largest dimension (or 0, 000 000 009 meters)!


Figure 1: a) I am grabbing a wand used to hold the loop which is zoomed in b)the actual loop can only be clearly seen under a microscope like in c) various styled and sized loops are used to fish protein crystals shown in d) protein crystals are made of billions of proteins arranged in an orderly fashion as the symbolized zoomed in e) each protein makes contact (direct or indirect) with its neighbor proteins sustaining this way the crystal form itself , f)
a 3-D protein structure; notice it is not a time-resolved “movie”, it’s simply an inspection of a 3-D model from various angles. [PDB ID: 4PB2]; protein images made using PyMol software.

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– What important milestone have you reached until now?

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.

 Each specific protein is different, hence each specific protein often requires diverse techniques/protocols to purify them; but they also frequently follow the same broad standard steps. I consider a group of milestones corresponding to the successful production and purification of different proteins for my project as important, as well as a satisfying understanding of the broad steps themselves, making me feel comfortable with any future similar procedures I would have to go through.

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– Might you please elaborate a bit on how can you go from a protein crystal to a structure?

            Once all the hard work of producing, purifying, and crystallizing proteins has been successful (something many projects struggle with) we can head to a synchrotron facility to do a diffraction experiment (Fig. 2a-b). Synchrotron facilities are circular buildings where electrons are accelerated to high speeds. As these electrons are bent round in a circle, they release energy in the form of a very bright light, X-rays. The X-rays are used to measure samples, in our case protein crystals.

            When the crystal is hit by this intense light, it diffracts some of the X-rays which are detected, generating a diffraction pattern (these are images with various spots where the X-rays hit). The crystal is rotated, and a new shot is taken, from a slightly different angle than before; we repeat this until we are satisfied with the coverage or the crystal was destroyed – intense X-rays really hurt!

            After many diffraction images, its time for data processing! Each spot in the hundreds of diffraction patterns is recorded as a 3-D coordinate. These data are then used to “go one step back” (Fig. 2c), we create theoretical models and calculate how close they are to the real protein. After this comparison (theoretical to real), we slightly improve the model, and by repeating this cycle over and over, we get a better and better theoretical model, until a point where the model is so close to reality that we cannot see any significant difference. Its like a guessing game, where each guess is closer and closer to whatever we are trying to guess, until we find the best solution. In the end we get a very credible 3-D model of the protein, at which point, a bottle of champagne is opened! After all, scientists also party 🙂

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

            Of course, I’ve visited 4 different participating labs and in every case the professors and students have been welcoming and helpful.  I can say I have not only learned a great deal by belonging to this network, but also met many different professionals with whom I am able to continuously exchange knowledge while progressing my project.

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

            I wouldn’t recommend it to everyone, working abroad and in such a network requires the student to be very versatile. The person needs to be capable of both moving to another country to start their project and moving a great deal within the network participating labs, sometimes feeling somewhat like a scientific nomad!

            Having said this, I think that anyone who can handle these requirements should certainly grab this unique opportunity. The position provides the student with such a significative dive into the projects’ network, that I think is almost unrivaled for other PhD projects. We have the opportunity to meet a lot of professionals, with a variety of scientific knowledge that really shows how no human can really understand all of the ongoing science, but simultaneously how a collection of professionals can work together covering every (hopefully) knowledge gap the surrounding colleagues might have.