 So, good morning everyone. And yes, so not much has been done with the membrane proteins in your frontal sclerophy. So, I will be talking briefly about it today. So, why we really want to do neutron sclerophy? So, as we know that we cannot study hydrogens with X-rays due to their inherent properties of lower electron density. It's not visible in X-rays. However, there are some X-ray structures at subatomic resolutions where you can be, you can see hydrogens but not as clearly as in neutrons. If you see in this image, this is an X-ray structure of a soluble protein where there is not like very clean electron density around the hydrogens. But if you see in the neutron structure, it's like such a beautiful density around the hydrogens. So, this is what we try to find and this is really important, especially when it comes to enzymatic mechanisms or rados-driven mechanisms in any of the systems. So, that is really important and that is what we can study with the neutron crystallography. And if we go to the PDB, currently there are like very few neutron structures with none of the membrane protein structures deposited so far. However, there had been like few neutron structures like studied but they are not deposited of membrane proteins. There are also few bottlenecks with neutron crystallography, firstly the weak flux of neutron beam due to which the problem comes of growing very large crystals. And of course, this is major problem with membrane proteins because we always have issue of having enough protein to even go for X-ray crystallography. Another problem is negative coherent statin of hydrogen which creates background noise and also electron density cancellation. As you see here, if we exchange this hydrogen with its counterpart, the deuterium, then you will see in this figure that in the sample environment, we exchange H2O2D2O and slowly the covalent diffraction peaks were like visible with time. So, that is what we also try to do when we grow crystals for neutron crystallography and we exchange hydrogen to deuterium. And another major advantage of deuterium ladian is you can even get away with like smaller crystals for neutron diffraction which will be possible when ESS will be running. So, ladley can be done in like three ways where you just simply dilute your protein with deuterium or you produce the protein initially in the presence of deuterium that I will be discussing further. For this study, we have to choose a protein which is like very well studied and highly stable because neutron crystallography is a complementary approach. So, we chose a OMPEF as the modelling membrane protein system for this study and the best extra structure salt is at one point angstrom resolution which is pretty good for us to begin with. Also, Ali has already mentioned that in his previous papers as well. So, with the help of neutron crystallography, we can study the how the membrane proteins are actually like intact with the detergent or in the membrane surface. So, it will be really interesting to see in future if we can do it with neutron crystallography. But in terms of OMPEF, we want to understand the like flexibility of the water channel which involves like hydrogen network of many acidic and basic residues which can be further used to kind of design the pore of this membrane protein which has a potential role in drug resistance of bacteria. So, what are the current strategies to produce deuterium label protein and what we did in our study? I will be discussing here briefly. So, in the current strategy, it is a very time-consuming process because you inoculate your bacterial culture on LB media. Then you have then you move it to another LB culture where initially you have glucose as the carbon source but generally we use in like for now the preferred carbon source is glycerol because deuterium label glucose is very expensive. So, to reduce the cost of the process, we generally prefer glycerol. So, then the bacterial colonies are adapted with the LB media with glycerol and this is further moved to a slam culture. After adapting following this process two or three times, we then move these we inoculate these colonies to a small culture where we have a deuterated minimal media with deuterated glycerol as the carbon source and we continuously repeat this process like for at least six times so that the growth rate of the bacteria is increased in the deuterium media and finally we re-inoculate this culture in a 150 ml deuterated media which is used as a starter culture for higher density culture or the large culture. Currently, for most of the studies, E. coli is the preferred host because as we already discussed yesterday also like because of its easy genetic manipulation and yeah so in keeping all these points in mind, we tried to improve this method of production of deuterium label proteins where one of the another PhD student like who is a post-op now so he designed and evolved strain which has a better growth in deuterium. So I transformed the ompeth plasmid in that strain and then from there we started purification which means we skip all the initial steps of adaptation and we directly switched to this step where we like inoculate the colonies and we start the initial culture. But in order to also reduce the hydrogen transfer in the cells, we grew the cells in hydrogenated minimal media and then to give them less shock, we first made a deuterated minimal media and added hydrogenated glycerol instead of deuterated glycerol and we let it grow over time and from here we measured the like optimum audages as generally we inoculate at like od 600 of 0.1 so we like centrifuge the like optimum amount of cells and then we inoculate the bigger culture with the pellet instead of directly inoculating with the liquid. This reduced the amount of transfer of hydrogen in the media. All the purification of the of all the deuterated proteins is done in hydrogenated buffers and then in the end we do HD buffer exchange with the micron filters for the crystallization setup. I have to mention here that in this study we use three different carbon sources to compare the yields and protein stability of OMPF so we produced protein in presence of hydrogenated glycerol deuterated glycerol and also we use rich media deuterated algae as the carbon source. There are few like shortcomings in this process firstly wish it's always preferred like to use kinamide shenamycin resistance plasmid because you keep the cells for long incubation so MPC lean resistance might lead to leakage so we should avoid it and also we should prefer T7 RNA polymerase as the expression. So initially it's really important to do expression trials for your protein as the process is quite expensive so it's really necessary and also it's it can be very time consuming if the cells go in lack-face so it's really necessary to start with an initial expression test and know where your cells reach the optimum ODI or what is the best optimum ODI for your cells to be induced and later on harvested. So we did this short study and we compared all the all the cells grown in different medium with different carbon sources so as you see here that deuterated media took like 17 hours to reach optimum ODI of one where we usually induce the cells as compared to hydrogenated media which took like nine hours but this can sometimes change if there is a long lack phase so this is really important to study. Moreover we also optimize other factors like if we start the culture at higher ODI instead of 0.1 we realize we get higher protein yield and optimum IPTG of course to get maximum protein yield because you also get protein yield is also reduced in deuterated media so it's good to optimize as much as you can to get the highest protein yield. So here we purified so once we get the optimum conditions we purified we produce a protein in large culture and then we purified it so here we see the purified bands for all the proteins from different media and the growth was quite comparable because from hydrogenated media we got like one mix per liter and which was also comparable to the deuterated counterpart and here I mean which is not like this yield is like quite good for a membrane protein because usually we don't get such high yields in membrane proteins and then to confirm that how much was the deuteration level in the protein and if it was really deuterated we did mass spectrometry and compared the difference in mass to calculate the deuteration level and as you can see that our protein was almost 100% deuterated when it was and all the components in the media were in deuterium and when they used deuterated algae as the carbon source the protein was like 99% deuterated. We further as have to go for structural studies so we also wanted to know the homogeneity and the stability of the samples so we did some thermal stability assay by using manual DSF and we found that deuterated protein is more stable than the hydrogenated protein which was expected because when the protein is when the protein is in deuterium then and it is just as the XD exchanged then the hydrogen bonds are more stable which makes it more stable and that is why the thermal unfolding was at higher temperature for the deuterated protein than the hydrogenated protein. Also since OMPEF is a trimeric protein it was interesting to see that the trimeric form was like unfolding at higher temperature instead of like monomeric form being stable until like until the end and this has been reported previously also with OMPEF from other organisms but not from E. coli so this was an interesting thing to know about the oligomeric oligomeric structure of OMPEF. So in order to grow large crystals we use many techniques like vapor diffusion, micro dialysis, batch and highlight and capillary counter diffusion but we got like so far the largest crystals in two techniques like capillary counter diffusion and vapor diffusion where in capillary counter diffusion we could go up to 150 micrometer I think this can be still improved I would say and this has been improved with another membrane protein circa done by another student in our network but for vapor diffusion in hydrogenated we could go up to 1.2 millimeter however these crystals did not like diffracted at room temperature which is the major requirement for neutron crystallography so we will be using a cryo stream at neutron instrument to diffract these crystals and when we simply hd exchange to like we exchange a reservoir solution in the wells of vapor diffusion we could get the crystals up to 400 micrometer size so once you optimize the conditions for crystallization in hydrogenated buffers only then it's nice idea to move to deuterated conditions and optimize it. Generally the conditions should be same but since the solubility of the protein is like affected in deuterated media it's nice to re-screen at least around the conditions where you got that this is in hydrogenated buffers so as you see that in the hydrogenated when we use hydrogenated minimal media we got like pretty nice crystals as we see for onpef but in the same conditions we did not get like as pretty crystals at hydrogenated one however these are kind of the first crystals I have from deuterated samples so far and I will be diffracting them like in this week but we have a nice diffraction from the protein purified from hydrogenated media. So to conclude I think this protocol is pretty much good to go with if you want to purify a polluted membrane proteins just in your lab without using any large fermenters and we also observe that the deuteration has like also affects the physical chemical properties of proteins and this is kind of a challenge because then you have to maybe find you the crystallization conditions in deuterated buffers and sometimes it can be tricky. Moreover deuteration also affects the secondary structure motifs and hydropobic surface exposure during the unfolding event so we can even look into it into its future and these studies can also help us to study the interaction of protein over the like with the detergents. The major limitations on neutron crystallography which is its large crystal size and small units and volumes which which will not be whole once we once ESS is running because of its high neutron reflux. So I would like to at last thank you my supervisor Escox-Hannon and class 1.5 in whose lab I work in the university Zoe Fisher from ESS from time to time helping me out and Vinadis for designing the stream Maria and Celest from LV3 for sometimes doing remote data collection for my crystals, last scale facilities ILL and Maxport and Professor Paul Neeson for allowed me to do some work on capillary counter diffusion in his lab and my thesis committee members and whole ram computer. In the last I would like to suggest you to listen to this phone because it's quite interesting and the it's a very cute song about electrons and neutrons. Yeah thank you and I'm open for questions. Thank you so much Wati. That's brave to take on such a challenging project but that's very good of you. So we're a little bit late but I'll just take one question here at least. So have you considered macroseding of the OMPF? Yes I didn't have time to show many things but I tried micro seeding, micro seeding. Micro seeding worked pretty well but macro seeding was not, I was not very successful with macro seeding I would say. I had issues to stabilize the membrane protein every time whenever I did that.