 Thank you very much for the invitation. I'm really glad to be and flattered to be invited and to share some of our thoughts with you. I also have to say I come here with I would say two hats. One as a head of the EMBL gremobri unit which I took in the middle of last year and the other one as a scientist and so my first two slides are actually going to be related to the fact that with the first hat and that is head of EMBL gremobri and just for those who might not know the details of the EMBL gremobri site I would like to just mention that EMBL gremobri is part of European Photon and Neutral Science Campus which is in turn composed of IBS, the French Institute of Photon and Neutral Science Campus which is of course a very big scientific hub but in particular from my point of view really brings together a critical map of different kinds of structural biologists. So I believe that we are nowadays really in a golden age for structural biology and in particular when we think about these what I just showed you before here we are actually in a really blessed position to be on the same side together with the brightest neutron source on the planet Earth until ESS is going to be in function as the brightest synchrotron source on the planet Earth. Together with what I said the Bifrostructural Biology community on the top of that we have of course the known resolution revolution caused by developments in cryo little microscopy we have gene engineering CRISPR cast capabilities which are particularly important for combination if with in-situ structural analysis we have alpha fold and of course all these allows us to really do integrative structural biology approaches. Being part of the NBL also brings a different I would say important aspect like being really part of a big institution involved in molecular life science research which also offers a lot of scientific services platforms to support this research and being part of this new research program from molecules to ecosystems. Finally I would also like to mention that the NBL Grenoble is not only a place for where we do of course science but also we do technological and technological developments of instrumentation for not only diffraction that of course started with a diffraction but also for nowadays cryo little microscopy and x-ray imaging and also crystallization of robotics. Going back to what Trevor said in the slight historical touch the NBL Grenoble site was funded back in 75 that means that in two years we have 15 years of existence over the site and that was funded there because ILL was there and the management there thought that this is really this was a very forward-looking decision to put aside the data that can capitalize on the best neutron source available to do structural research research involved with structural biology. A few years later similar thing happened in Hamburg where the Hamburg site was funded because of the synchrotron there and actually the first research was actually as Trevor said propelled by research in the field of processing by Ken Holmes. So from now on I will now switch to more research oriented talk and that is going to like to talk about the work we do on muscle and I would like to first remind us all this is textbook science that muscle as an organ is composed of single muscle cells or muscle fibers which are internal composed of myoclibrins which are internal composed of sarcomeres which are the building blocks of the basic contractile units of serrated muscle. This crystallographer would say this is a unit cell. Now you have seen this kind of a representation of basic filaments of muscle where you have acting filament myosin sliding which the two of them results in a contraction but you also see here tychin which is the longest known protein it spans from the beginning of the sarcomere to the middle of the sarcomere is some sort of a regular ruler and the blueprint for it assembly but we are not actually interested in the contractile machinery but rather in this area here which is called the Z disk which is the boundary between two contractile units which is sarcomeres this is where filaments in particular in acting filaments from adjacent sarcomere is actually linked together by these majors in disk prompting point alpha. So linking filaments is an important because if everything were moving you of course wouldn't get anywhere only filaments that are gotten contraction are need to be fixed or furthermore these disks also allow for traditional force from one sarcomere to another in order to actually achieve the macroscopic contraction. So here is schematic representation of the Z disk by now the current directory of proteins is somewhere around 50-60 what I'm showing here is only the major the major players. So if you look at the sarcomere in the let's say standard way you see these variations that's where the striated muscle perm comes from this results from different densities of proteins along the sarcomere and you can see the Z disk is the darkest meaning the highest protein density if you do the transversal section through the Z disk what you will see with this thematically presented here is basically you can see that this is basically a para crystalline highly ordered structure which displays a tetragonal symmetry where one active filament from one sarcomere is surrounded by four active filaments from adjacent sarcomere and this material connecting them is alpha active. So I find it every time I think about it really fantastic and amazing that in your resting muscle now you have this para crystalline highly ordered organization of filaments in the under contraction this is a bit changed but highly ordered. So this architecture has recently been seen also with cryosome tomography in cardiac or striated what I said skeletal muscles and you can see again active filament surrounded and connected by alpha in the tetragonal shape. Though these are pretty recent reconstructions that's about 10 to 20 oxygen. So a long-term goal of my research is to understand what is the molecular architecture and assembly of Z disk from muscle Z disk protein. How do they assemble what the hierarchy of assembly? How does this how do these proteins then basically come together from quite this ordered initial assemblies called Z bodies or this ordered contact into something that is quite ordered and also mechanostable and how is then the function and the force actually transmitted from one sarcomere to another. What you are also looking at but I will not talk about because of the time means how do different disease mutations actually disrupt this finding to the assembly. You of course know there's a lot of cardiobiopasies and biopasies that are caused by mutations in the muscle proteins. So there's already some such information known about the different bits and pieces of proteins and some complexes under the risk but there is still a long way to go and the way we are trying to address disease by integrative approaches combining different structural biology approaches molecular biophysics and biochemistry on repostituted complexes and we are now trying to go also in the direction of more C2 research. So the first thing I would like to talk about which is the major Z disk protein connecting anti-parallel active filaments in the Z disk. So if you put alpha-actin in close to actin filaments what will happen is that alpha-actin will cross link actin filaments as you can see here all along their legs. This does not happen in muscle you can see actin filaments only cross link by actually only the Z disk and that is because alpha-actin is actually targeted to the Z disk to show specific interactions regulated by PIP2 and these interactions have to do with interactions of alpha-actin with titin is the longest protein under the blueprint of muscle assembly differential before. So as you can you can see here alpha-actin is an anti-parallel dimer. It contains actin binding domains of the two extremes which makes sense as an architecture that it can cross link actin filaments here. It is followed here we have four specter-like repeats and that the two extremes we have a calmodulin-like domain not have been a diskal-modulin-like domain does not bind calcium which has lost this capacity through evolution because contraction believe that that is because contraction is a calcium-regulated process and is being a structural protein does not want to be influenced by different concentrations of calcium but this calmodulin-like domain actually binds to the juxtapose subunit and to this connecting region called neck region connected by domain and the body the rod part. So the model of the regulation goes that PIP2 would bind to this polar actin-binding domain with this cataphobic chain which would disrupt this interaction deep calmodulin-like domain and the neck. This would open and this part would now be able to bind to titin to specific titin regions called Z-repeats in the Z-repeats and this interaction would therefore target alpha-actin directly to and only to the Z-repeats. So these Z-repeats can come in different numbers from 1 to 7 with more or less the higher or lower affinities particular 1, 3 and 7 have a particularly high affinities and if we look do the titin-salinatobe Z-repeats at this neck region you will see that there is some sequence chromology which also makes sense because it's something like the way either bind to the neck or binds to the Z-repeats and that's the reason also why this mechanism was termed both for liquid level regulated intramolecular pseudo-legal mechanism. In order to understand this mechanism at a more molecular level we first determine the structure of alpha-actin which you can see. Here this what you can see is a structure in the closed conformation acting by laying domains. Z-repeats, 3-heel expandals are moderately like a domain clamping on this neck region here, your neck region as the model was predicted. I always like to see that this rough region is usually like a pasta structure. Now we then designed two different types of mutants to actually investigate better this mechanism, proposed mechanism. One is we inserted mutations on the PRP2 binding site where we mutated this positively charged residues where the negatively charged polar group of PRP2 blocks and this therefore would be not activatable mutants. PRP2 not activatable mutants and in fact you can see that while the wild type binds PRP2 this mutant has a much reduced binding of PRP2. The other mutant that we designed was the opposite I would say is the constitutively active mutant where we inserted mutations in this neck region here in order to prevent this calmotting like domain from binding here in order to generate a constitutively open mutant which would therefore not meet PRP2 to bind to type. This mutant we called Neck Mutant because that's the way the sequence that we mutated right after that was. So we then first of all investigated the structure of this Neck Mutant is this really an open co-formation. We did that both this molecular x-ray schedule where we do can see that this Neck Mutant has an open co-formation with a calmotting like domain not docking on the neck anymore. We also used double ectone-ectone resonance in order to again confirm this open co-formation. We then used a lot of different binding assays where we saw that looked at the affinity of type in wild type. Neck Mutant has of course better affinity because it is open and this is comparable to the affinity of calmotting like domain on its own. And we also protect the affinity of this PRP2 mutant to type in in presence or absence of PRP2. So this type in PRP2 non-sensitive mutant of course has the same affinity as the wild type because it cannot bind PRP2. You also check to whether this mutant that we created still has retains its basic function which is binded to active filaments. That was done by acting with sedimentation assays and you can see that in the segment we obtained acting but also an alpha actin but also Z-repeat which means that this mutant not only does it bind active filaments but also brings it with itself the type in which is what it was expected to do bind at the same time active filaments and Z-repeat. Finally we also looked into cells in the cardiac muscle cells from that and we can see that if we transfect them with the nick mutant not to not see terribly well here but you might like to trust me that if we transfect with the nick mutant we really see a disarray of the organization of this strain variation which means of the assembly of the of the muscle and we also see a notable effect of the dynamics that means that if we transfect look at the dynamics of the nick mutant there's the one that is constantly open this is much more firmly bound fossilized on the Z-disc and we believe that that is because it cannot close anymore and therefore it is always radiant from there to bind to Z-disc compared to the alpha actin mutant. Finally we looked at acting filaments decorated with the acting binding domain this is some part of the reconstruction took then our alpha actin mutant and superimposed it this is now superpositioning powerpoint but what we did then is really superpositioned on both sides of acting binding domain and what we got is this and that's wrong right that's wrong because I said before that alpha actin can bind and cross link anti-parallel acting filaments there's no such thing as perpendicular acting filaments in muscle that is because or even if you let alone muscle even also if you put acting if you do this in vitro what you get is this bindings that is because if you look at alpha actin in the closed conformation and you look along the rod region for this part here you will see that there is a built-in twist in this rod by 90 degrees which means that this actin binding domain and this actin binding domain so you rotate it with it by 90 degrees with respect to each other so in this closed conformation we cannot actually achieve this bundling uh bundles formation so what we actually think happens is that PIP2 binds opens this conformation and therefore this neck region which is not docked by commodity like domain anymore unfolds and we know that this happens because we have NMR data on on this region as well alone and this therefore allows for acting binding domain to reorient in the way to be able actually then to cross link anti-parallel or the acting filaments so we believe that it's actually you need this structural flexibility to actually achieve the basic function which is crossing the might be a bit counter intuitive that we need structural flexibility but you need to achieve this rearrange structural re-automated structural gymnastics in order to make it work but at the same time acting filaments and dock and be connected to titin but the next question we then ask is what is actually the mechanics of alpha actin in titin interaction namely affinities of titin they repeat three of hands are low micro border forces in the muscle are about under construction about 200 people newton and the question is how is the task of firmly anchoring titin within the z-disc under applied forces actually so again i would just like to mention uh repeat again that we have this titin z-repeats to which alpha actin binds we have different isoforms of titin you have past muscles which have less titin repeat and therefore is stiller than this slower muscles which have more titins and z-repeats and sicker than this that means you have more or less alpha actin is bound in this originally discussed here so in order to understand the mechanics of this we actually use optical tweezers please be reminded here that when we think about force in muscle the force is transmitted in a long acting filaments a long time so we use optical tweezers where we took uh yes 3 3 4 is currently binding to terminal law which binds to titin made the camera and start pulling so what we saw is here the force propagates through we have hands and titin so what we saw is that at about uh force of about four people newton can see the destruction of this interaction and if we keep the force constant at about four people newton we see these events of partial unfolding or destruction of this interaction of the z-repeats to the hand and uh then total unfolding we could also determine affinities of these repeats to come out in like a domain by adding a peptide in the let's say reaction mixture and we got the thing so about five four micro molar we then did this for all repeats and we saw when chi affinity for repeat one three and seven as you can see here all about the low micro molar while the repeats uh two and five displayed weaker affinity similar as also the neck which is also obvious because the neck should have less affinity than the z-repeats after having done all this we actually realized that the design of the experiment was not ideal because we were as I said pulling here along uh yes hands and titin in fact we should be pulling only along titin therefore we redesigned the constructing order to really apply force only through the titin z-repeat and here what we got is that well before we saw we saw unfolding at forces of about three to four people newtons now we see these unbinding events at 12 people newtons and again if we see if we keep the force constant that what was this about 10 people newtons also again starts in these unbinding events also we determined again the binary affinity so putting this together is good as concepts how is now the task of firmly anchoring titin achieved we believe that uh we have a here a VDT at work so first we have to remember that we have forces on the single titin molecule which are of about five people newtons now if we look at this concept here which was where we were pulling along EFNs and they repeat here we have a midpoint force of about 3.5 people newtons that means this kind of a construction does not actually sustain the forces that are applied in the master but if we look at this design here we can actually when we pull along titin only we see that in this way this construct actually sustains forces of about 13 people newtons this means that the whole system actually evolved in the way to endure forces along the direction of active now we also have to remember that we have several kind of human titin binding sites one three and seven on the top of that we have to we have of course dynamics in the binding and unbinding so that means that in every time point we have more than one of these interactions taking place and of course we have here a VDT effect of three binding sites uh bindings the potential binding at the same time where of course interaction free energy sum up together uh and that of course means a this multiplier so we have therefore these dynamic bonds acting together to lead to a long term table anchor of titin of alpha teaming on the titin on the alpha team finally we also looked at another interaction between alpha teaming and titin and that is the interaction of this region called ZQ which is after following this example so as you said they could be this binary like domain but the ZQ was postulated to bind somewhere in this rock region here so there is this is a structure of interaction between the one like the main and the one that is actually basically this is a model which was generated 14 years ago but it was suggesting that a dimer of these ZQs would actually bind this rock region here so what we then want to try to understand is what is actually the molecular mechanism that uh drives this asymmetric sorting of alpha teaming in the as you can see if you imagine three alpha teaming is bound into the ZQ you will see that this middle one will be bound to the ZQ it's only while this outside ones would be bound to ZQ but also to this ZQ which is here in the so in order to understand these interactions we designed several different constructs to investigate the interaction between Z to be seven and Z to be like five so Z to be seven ZQ alone is behaves as the asymmetric disordered protein that you can understand from sensitive group of photography profiles from the tv as well as from the x-ray scattering experiments where you can see the factory plots that classically representing a neutral ZQ disordered protein now in order to understand better the teaming tv design a number of different constructs of alpha teaming and looked at how does this constructs bind to ZQ and we saw that the bindings as long as you have the roto domain the binding is there but if you cut the roto domain in half that means if you have the half-binder then your body's binding suggesting that you need this middle spectrum repeats two soon three for the binding to actually take place and in fact this we saw then starting the crystal structure of the rot in complex with ZQ where you can see that what we can actually see is only two linear motifs that bind to this central spectrum repeats two and three as we also hypothesized based on binding experiments we have to say that this experiment or the assignment of the structure to this electron density was quite an effort we actually made also some mutations also selenium metronome mucoussine we inserted here in order to then exploit the number of different Fourier in order to assign the sequence to the electron density now looking at the alpha fold prediction of this region here the Z-repeat seven and ZQ you can see that it is mostly this order but it predicts two helical elements one is the Z-repeat seven which i showed you from the structure that NMR structure that i mentioned before that actually adopts a helical structure when bound to some other rectiline and this helenium mucoussine this part here is you don't know yet what advice but one might have also advice to something that is when it is when it is bound now we also generate the family mutations in order to validate the this interaction with its timetable and alpha kinase we actually saw that we see abolish if in certain mutations we also abolish protocolization of alpha kinase and timetable then we asked ourselves is do you really need the entire ZQ motif or is it enough to only have the peptide that contains this nm1 and nm2 in fact we see that you do need also this flanking regions that we don't actually see electron density but they are obviously involved in assisting and counseling then we also asked ourselves is the length of the linker between the Z-repeat seven and ZQ actually important can i shorten it or can i lengthen it and modulate the affinity in fact we can but whatever we do we disrupt we make it worse we thought actually that well you could by directing we should actually be able to improve the affinity if we lengthen the linker but whatever we do we lengthen it or double it or fortune it we disrupt the affinity telling us again that it was really cost it's really customized and that these connecting regions are also involved in productive interactions finally in order to them understand what is the structure of the complex of the Z-repeat seven and Z-repeat combined crystallographic data together with smaller x-ray scattering data and used EOM so a sovereign optimization method to actually generate this what in IDP work is called the classic complex where you can see that typing actually remains apart from when bound to alpha key in a still in a disordered as a disorder problem now looking at this interaction between alpha key and Z-repeat the seven we actually saw that we have only one Z-repeat which is one Z-Q binding to alpha key although the alpha key is a diner and there would be potentially space for both and we believe that that actually is important in achieving this asymmetric sorting of alpha key where only one alpha key can be loaded by the Z-Q motif here meaning that actually alpha key or Z-Q regulates its own security in the in this interaction finally we wanted to see how does this structure look like and therefore we generated a construct containing two Z-repeats of alpha key of the type and Z-Q we put an MVP on one site in order to be able to see the orientation and therefore we obtained a dimer or dimers to alpha key means bound to two Z-repeats this is a rotary shadowing image basically what we were looking is at something like this without this intermediate alpha key here we have a low cryo-reamp reconstruction that I have to say we are still fighting with this of this construct we have everything or the troubles that you can have with this from preferred orientations to aggregations and everything but nevertheless what we can do is compare this reconstruction with the cryo-et reconstruction that I showed you at the beginning that where you can see these alpha key means cross-linking active filaments in this is a skeletal muscle in this and we can see that our construction actually nicely fits into the primary theoretical structure in the Z-repeats so we haven't still given up on this one but what we are what we can see now is that we have this alpha alpha key being decorated with a pattern and that the pattern itself is actually regulating its stoichiometry and asymmetric sorting of alpha key means now the last few slides I would like to spend on the last interaction with alpha key and that is with fat which is a intrinsically disordered protein fat stands for filament alpha key in phyletonic binding protein meaning we can have a number of binding particles so one of them is alpha key now fat is also an intrinsically disordered protein credited as fat and we have I must say agonized over the design of constructs in order to to obtain the genetic process that will not be agonized for years finally what we use is an erase the base approach that is actually developed by from Karolinska that is a I would say a poor man's version of a spring from their own heart and with this approach we actually then found two constructs that were workable while starting from position 92 till the end of that one going to 174 you have to say this is none of such none of this kind of would be designed if you were thinking this was really something that came from this brutal screen this ghetto actually got started started first we again confirmed that yes these are intrinsically disordered proteins using spherical x-ray scattering you can see here crafty plots and we can model these two constructs and a c-termic construct as ensembles more or less of more or less compact structures then we looked at the binding affinities we can see here that alpha kimi binds to fats and I'm talking about c-term of delta 91 region by a two event nano-molar and low micromolar affinities you can abolish this interaction by designing first and the mutations you can also see that path dimer of alpha kimi at the difference to z2 here does bind to fats now analyzing the energetics of this interaction you can see that it is an entirely driven which means that the interactions that dominate this complex formation are polar and I will come to this later then we then we wanted to use an NMR to assign and map the binding sites of alpha kimi to fats this is the c-terminal region of fats when we add alpha kimi we see the depletion of a number of signals and that is those that are involved in binding therefore we have an overall signal because there's something we're doing getting done this we wanted then to assign alpha kimi to fats unfortunately we didn't actually manage to assign exactly the most interesting region which is the one where the binding actually happens and you can see that you have the biggest effect of the intensity of the signals in this region that is here but we would not get the residue by residue assignment so at this point then I managed to convince somebody in the left to crystallize this complex because everybody saw that she's insane because she wanted one complex so we then did the disorder protein we actually bought three different complexes but again seeing fats interacting through linear motifs two short linear motifs all the rest this was a construct was about 200 aminesis was not seen in the electron density therefore we again use molecular x-ray scattering to generate the structural information on the complex to see what the rest of fats is doing and again the model is using EOM this is now an integrative model of alpha kimi decorated with two molecules of fats and you can see again this is a fatty complex with alpha kimi decorated on one side of its slightly concave surface rot surface with fats so I will skip this for the rest of time but if we now think about the architecture and alpha kimi in muscle and decorated with fats which is a hub for protein interactions we can see that fat is actually stemming in a certain direction in the viscose and therefore is attracting in certain place in certain directions the other biodegradable the other proteins we can find therefore being really an organiser of this viscose naturally what I'm showing you here is I'm pretty sure not the way it looks in the same with muscle because this many of these parts will actually be loaded with other proteins that will not be physically disordered anymore but what I would like to show here is that these are the docking sites for other proteins to assemble now in the course of working with alpha with fats we actually saw that it actually forms lipid condensate especially the serum region actually forms this macromolecular condensate which was I would say really a serendipity we were we ran for fun the sequence through this predictor of lipid phase separation which got a very high score in this region here which is also the region which is involved in binding to alpha kimi and we we looked under the microscope and we saw this phase separation we then naturally after ourselves that alpha kimi go in these droplets and it does go as you can see here with well actualisation and you can also see that if you wrap it you will see that alpha kimi actually easily plan with state exchange because of the biophilium that it can actually penetrate this mass that is formed in lipid condensate we also saw that if we increase the concentration of alpha kimi we can actually dissolve these droplets so there is some mechanism over the solution of these droplets with increasing concentrations of alpha kimi which also makes some sense because this phase separating region and alpha kimi binding will be in part co-occurrence so why is this at all important because we all know that it's those who have crystallised proteins have seen more condensate than crystals phase separation than crystals even this could be important because it's known that sarcomere biogenesis is a hierarchical process that starts from what is called Z-bodies these are five types that merge together to form Z-mix mature Z-mix and in this five types the major components are alpha kimi and 5 which is the ones that are actually made before and the couple of other Z-disc proteins that are also then present in the mature Z-mix so could it therefore be that these Z-bodies are macro molecular condensate I think this is of less this is a site of site importers more important to my opinion is the question how does an order paracrystalline system formed from this this order partially this order the bodies would it be that these parts together with other some other process is actually some sort of a hub for protein protein tractors bringing the major players together and then of course once they are brought together can we actually disassemble how does it disassemble disassemble because we have different concentrations of players taking place during the sarcomere biogenesis in fact alpha kimi concentrations increase during the sarcomere biogenesis in order to then lead to an order start so we have we are of course now trying to play more with this I think this is a Pandora box that we opened and what we have done so far is we have actually reconstituted this condensate with four out of six big body veins and body components if you add alpha kimi active with first of the stream effect which where the stream I think we must be at him and as we speak we are doing now the same adding also here we see so of course what we are doing now is on the one hand using the structural cell biology the structural biology two on the one hand in vitro the system the system looking at phase diagrams hierarchical interactions so of course looking at connected complexes but also doing this in cells and the system we are would like to use here is and we are actually designing cell analysis stem cells that are myosides in particular there is a model which arrests at the state of the same points this is a proponent knockout which can be inducible so one could actually look at the system arrested at the same person then promote it into the more mature state because we can what we're using we'll be using here is it was fluorescent properties looking at the discoloration properties of these eight bodies but also for solution microscopy and dynamic studies like that and I believe that this is also a good material for the process perhaps also for so if I have two more minutes I will just show one let's say exercise that we are performing as we speak and that is using alpha alpha fold to actually predict now the interactions of the zebras proteins we know that there are about 60 proteins so for the moment we selected 13 16 different phase because some are dimers or heterodimers and we chop them into domains so that we were actually doing pull down with about 90 different minorities so and we did pull down everything against everything this is still in working in calculating you can we got quite a complex interaction diagram but if we now I would just give you an example of one we looked at of course our known alpha we could see that we could nicely predict some interactions in alpha fold that we know are there and we are spot on like with typing like with the last binding to the in this a domain of I think that is a domain of us it was so nicely predicted some interactions with for example my opinion but it was also predicted some novel interactions which are of course now good substrate for say further experimental validation another example that I would like to show here is a nice correct prediction of the interaction of my opinion with alpha opinion so this is the direction of the domains only if we now look at the prediction of the full my opinion which is composed of two iG domains and the rest is in this order you can see that iG domains are of course okay but if you think with this structure here it is not that it comes like unnatural in effect it is unnatural because this is the prediction this is actually the tax reconstruction of full length my opinion which we did using again EOM as the model tool as a way to model the ensemble again interaction was predicted correctly but the whole structure is of course it's known that alpha fold cannot do the dynamic dynamic systems yet of course what we are doing now is doing this alpha fold down also with higher order only commerce and templates like a filament or dimers tetradimers using therefore structural templates so with this I will stop here and I would like to thank my current colleagues my past colleagues funding and I would like to give you my saying that by this African saying that says if you want to go fast go alone if you want to go far go in company this is the company and from here that was the that's the day of here but we're doing it on the 31st of January this year thank you very much