 I am from Delhi Technology University that is in Delhi and here I am working with Dr. John Makhle for the past two months and this project that I have been working on is investigating the interactions of bacteria and C-vibers with other matter modules in the ion sulphur cluster biogenesis. So I work through the interaction A, workflow, results, conclusion and the summary. Now what is an ion sulphur cluster? Ion sulphur cluster are basically ensembles of the ion and sulphide centres. They are found in a variety of metal proteins like the ferroloxins, the NADHD hydrogenary and the most common form is the 2FE2S form which exists as a rhombic structure as shown. Now it can either be in the reduced form or the oxidized form. The reduced form has one ion as in the 2 plus and the other in the 3 plus A while in the oxidized form both of them are in the 3 plus A, the A. Ion sulphur clusters are ubiquitous and are required to sustain the fundamental light processes. They participate in many biochemical processes like the electron transfers, substrate binding, analysis, regulation, sensing etc. But the biogenesis is still a mystery. As in a lot of groups are working on this particular thing but then again controversy still exists about the action mechanism and the cascade of interactions that are taking place between the proteins. So the three mechanisms that are suggested are the NIF which takes place in the nitrogenary and the IFC which is the ion sulphur cluster and this is the one which I have focused on and the third one being the sulphur-utilizing factor. Now this takes place only in cells which undergo oxidative stress. So this is the basic mechanism which takes place in a eukaryotic cell. This is the mitochondrion. This EBO-ISU1 is the scaffold protein onto which the ion sulphur cluster is then it takes place. Now it is known as ISU in prokaryotes and the other partners like the NFS1 is the NFS16 these are the arrays. It degrades cysteine into anony. So it has to be released sulphur which is assembled on the scaffold protein and on the other hand it acts which is acting as an ion shaft protein. It brings the ion and assembles again onto the scaffold protein so that the ion sulphur cluster is formed. This is the first step. Now the second step, the ion sulphur cluster is transferred onto the protein with the help of certain molecular shafts like SSQ1 and JAG1. The third axis is the eukaryotic ortho-log of CYIY which is a protein I have concentrated on in my project. This is a very controversial protein because many people say that it acts as an ion shaft protein just as I said. But then other people say that it is a gatekeeper which helps regulate the amount of ion sulphur clusters that are produced according to the number of target proteins available. And then some people say that it is not an ion shaft one at all and ICA which is another protein. It is actually responsible for bringing the ion and assembling it onto the scaffold protein. So this is a very controversial protein and that is why it is very interesting to study it. The workflow that is what we did. Firstly the NHSQC of CYIY then with unlabeled ICS, then with ISCU and all treatment together. Then we repeated the experiments in the presence of ion-3, then ion-2 and then gel filtration studies of that and then data analysis. We preserved all the files using NMR-python in the part used. Now HSQC. HSQC basically stands for heteronuclear single quantum coherence. There is basically a 2D NMR experiment. There is a 2D NMR so whenever you want to go in for experimentation and studies, for example for ligand and substrate binding interactions like we are doing. So this is the first experiment that needs to be done before anything else. So that is what we did. It needs to be performed using heteronuclear that can be either carbon 13 or N15. We used N15 for other studies and then the peak for each union proton attached to the heteronuclear appears in the spectrum. And the number of peaks in the spectrum should match the number of rescues in the protein but then if your residue contains nitrogen in the side genes then additional peaks do appear in the spectrum. So this is just another representation. This is the hydrogen attached to the nitrogen. And this is another one. Both of them appear as crossbeaks in the spectrum. This is what an HSQC looks like. On one dimension you have the nitrogen, chemical shade. And on the other you have the hydrogen. And the results. This is the NHSQC of Seva ever. This is a very beautiful spectrum as you can see that almost all the peaks are clearly separated and they are circular. So it's a nice spectrum. And you can see that our label the rescues. Now for late being for identify which residue is present where you need a whole lot of other experiments like the backbone and side-chain assignments. But we did not do that but I just went to BMRP and the chemical shade beta that was positive with it. I just hopped onto my spectrum and I was lucky to like obtain about the rescues identified. This shows an overlay of the Seva ever with ICS on to Seva ever. So you can see that some of the peaks that like the blue the blue peaks they have disappeared and only the red ones disappeared. And there is some shifted as a zoom into some of the rescues you can see. So this clearly represents that there is some interaction that is happening because some peaks have disappeared. Some have shifted so it shows possible interaction. So here this is the X-ray crystal structure of the Seva ever and the rescues in the other ones which are actually showing interaction with the ICS. This is an overlay of Seva ever with ICU that is also connected to Seva ever. So it is clear that all the peaks have completely overlapped. So it shows that no interaction exists between Seva ever and ICU. This result shows that Seva ever does interact with ion free as you can see there are a lot of shifts occurring and some peaks have disappeared also. I will just show you another picture. So I will just put this picture so as to show how the peak can shift. Here as you can see as the increase in amount of ion the peak has shifted. So it is clearly visible how it shifts and you can see this is the movement of this. Again I have represented the rescues in green as the ones which are found to be most affected by radiation of ion free. This is a chemical perturbation graph that I have spotted and the rescues which are represented in red are the ones which show disappearance of their peaks after the titration. And the rescues which are not showing anything are either because I do not identify them because I could identify 80% of the rescues and the some rescues are the peaks as we did not appear in the spectrum so that is why it is interesting. And here you can see the highest. This is corresponding to 1728 and this is one of the rescues four basic rescues which we also identified in this one. So here you can see that as you store you that Seva ever does not interact with ISE but then we can observe interaction between Seva and ISE is the values of ion free. Four rescues this here, this here as I have marked in here the four rescues which showed interaction. There is some subtle changes and the peaks are occurring on the addition of ion free so it shows possible interaction between Seva and ISE with the help of ion. ISE studies were done in the anaerobic addition and we were able to reproduce almost the same results as we are playing with ion free. The same rescues showed changes as I had just shown you earlier. And then we wanted to just check whether the ternary complex is formed between the three with Seva ever ISE and ISEs. So we carried out the gel filtration study and a single peak was observed so it shows that the complex is formed and it was renewed at 70 mm. Then we also obtained some issues results and we need further experimentation to show what they actually need. Then we obtained similar elution volume of ISEs and ISE complex as well as Seva ever ISEs and ISE but there should be some difference which is Seva ever is around 10 kg. So it should have been resolved then we just have almost similar elution volume. And the second is the failure to isolate ISE ever and ISEs complex. So in the gel filtration result we just obtained two separate peaks for Seva and ISEs though we should have obtained just one for the complex because it has approximately the same affinity as that of ISE or ISEs. So that was again a very weird result. So we need further experimentation to see what it actually means. So yeah we have observed that these both ISEs play a key role in the interaction with Seva ever but I do and I am free both. And it is also clear that Seva and ISEs and ISEs have a definite role in the formation of ISEs but we also need some further experiments so as to clarify, so as to verify that ISEs as the ones we obtained are true and side directed metagenesis would help us in that because then we can just replace those residues with ironene and then study if there is any effect. And also for deterioration experiments to be done so as to obtain the peaks clearly on the spectrum and then iron fire them for me. To summarize, ISEs are central to life and important for a wide area of processes. The cascade of interactions between the various components of the iron-sulfur cluster synthesis is not that well established. There are still very huge controversies interesting and there are interactions between Seva ever, ISEs and ISEU only in the presence of iron and with iron that is. Now I would like to know if you know the Qur'an program, Dr. John Markley for allowing me to work in ISEU who is my co-writer and help me for the my entire ISEU. CHI help me in the beginning to learn everything. I used to bug him all the time and then real and they helped me through and for the finding which is provided by the University of Wisconsin ISEU, I would like to thank you.