Illuminating Viruses: Synchrotron Methods in Virology with Marjolein Thunnissen

Okay, so it's 1:00 maybe we start. Um, welcome to uh today's seminar. Um, it's my absolute pleasure today to introduce myoline and tisen to you. Um, I met her in March uh when I've been visiting the university um of Lunt at the MX 4. She will probably present to you and I was really impressed about what the Max 4 um offers. So um Mayoline is the senior science advisor of the MEX 4 and who doesn't know yet what the MAX 4 means. So this is an Swedish national laboratory uh laboratory providing um scientists with um X-rays for their research in collaboration with the L University and as I told you it's really impressive. I think you have 16 beam lines on uh running and there's even capacity for 12 more I read in the internet. I'm not sure whether that's [laughter] correct but I found it there. So um by my line she um studied um chemistry at the kingan university in the Netherlands and she also received her um doctoral um degree in chemistry at this university and already as an um undergraduate she became fascinated um by unraveling the complex complex structure um function relationship um of proteins using X-rays and myoline has um solved several protein structures. She has been a posttock at different universities for example the Glasgow University. Um she has been a postto at Croningan University and also at the um department of molecular biology at the Stockholm University where she then also became an um associated um professor. In 2001, myoline um came to Lund um to coordinate um the construction and the development of a specialized protein crystalallography beam line at the MEX4 lab and then since then she has been the science coordinator for structural biology at the MEX4 and associate professor at the MEX for Lund University. She also had been the head of the user office of the MEX 4 and the life science director of the MEX 4. And currently my is the senior science advisor at the MEX 4. And this means she's involved in coordinating the activities between um uh researchers, scientists and and the MEX 4. This involves a lot a broad spectrum of applications including protein crystalallography or cutting edge imaging techniques, small angle X-ray scattering and I'm sure you will hear more about it in the next minutes. So Marioan, thank you very much for taking the time today um to give us an insight into the world of synretron methods and for providing us with food for new ideas um how to implement this um techniques in our research and hopefully also start some um exciting collaborations in the future. So I'm very happy that you're here today and the stage is yours. >> Thank you very much Ava for this very kind introduction. Um the lecture I will give is actually it's going to be an introduction lecture and I'm going to both highlight what synretrons are and then I go into what synretrons have meant for fology. It will be a little bit historic, a little bit of an overview, not very deep and really an introduction in in what we can do and hopefully followup events at Panda will actually go much deeper into into the u the matters at hand. So let me start sharing. So share and now I just have to start the presentation and I think this should work because we tested it. >> Mhm. Yep. >> Um so um my lecture is called eliminating viruses in control methods for viology and I I thought because this is really the first uh in a webinar series organized by panda. Panda is a theme at the links institute. I thought I would make it really really an introduction into basically the heart of what panda is doing. So we're looking at uh viruses understanding the virus and fighting the virus as well as complimentary methods all to actually understand viruses more by the use of synretrons and neutron facilities. So as um Ava already said um I'm actually part of Max 4 and this is an area of Max 4 at the end stages of when it was being built. Um it's hosted by Lun University but it is also a national laboratory. So it's a Swedish national laboratory and in that sense it's run by both Lun University and the National Science Council. Um I will talk at the very end of my talk a little bit about how you can get beam time at Max 4. So I'm not going to go into that. I'm just going to say that we have currently about 300 staff members and we are the first what is called MBA or multiband acromat based synretron. And what that actually has meant is that we have gone from a facility where you can take photos of material to a facility where you can make movies of material and this is very very broad but it's a huge step forward and you need a lot more x-rays to do that and that is what this technology has brought us. But let me start from the very basics um to what we actually provide to our users and that is light. And um we we all know what light is but I I still want to go through this because I think there are some concepts where I think it's good to to to know what we're talking about. So as we all know light is electromagnetic radiation and it is formed of waves of different wavelengths. Um we we can see light the the visible light but there is also light that we cannot see and that light is especially important for work at at synretrons and this light can be infrared ultraviolet x-rays and gamma radiation. So if we look at the spectrum of light the electromagnetic spectrum and we go in the wavelengths you can see that and I for for very long wavelengths radio waves you those we don't produce at the synretron but what we produce at the synretron it's actually from infrared all the way to gamma radiation and there is this relationship that longer wavelengths light um has a lower frequency or a lower energy while shorter wavelengths have a higher frequency and a higher energy. And this comes back quite a lot because we talk about hard and soft X-rays. We can also look at it a little bit like in this picture. And this all picture also highlights why we want to use different types of lights because with different types of light we can actually study differently sized objects. So if we go to hard X-rays which are around 10 the minus 10 we really really can look at atoms. Well, if we go to longer wavelengths, let's say 10^ the minus 7, we can see viruses. So depending on what you want to study, you kind of pick and choose. Um, and uh this becomes very important at the synretron because we have the capability to do that. Now X-rays are very important and we know already that from the very very start that X-rays were uh discovered um because with X-rays you can see the invisible and this was shown from the very very moment almost the very moment that found I discovered X-rays. So this is the famous hand of his wife um one of the first X-rays that that has been made. Um and and this really um shows that you can look through material and and see things that you otherwise wouldn't be able to see. Of course, this was 150 years ago. Nowadays, we have different methods to look at the invisible. So, if you work with X-rays, we actually use three different methods um broadly speaking. So the the first one is imaging which is basically still the same kind of idea as run's hand uh but we can go in much more detail and we can go to the nanometer or beyond level to look at um to look at material and to understand organization of matter and matter can be on a cellular level. It can be tissues. It can can be all kind of different things. Um very many experiments use scattering and defraction. Um this is my field. Um so I will talk mostly about this later on in my talk. Uh and scattering and defraction really um allows you to see details on at atomic level. And then finally you have spectroscopy um which allows you to understand the electronic structure of materials and the chemical state. So these are the three main methods that are being used and I will show examples of all three. So now what is a synretron? So a synretron is basically an accelerator of electrons and these electrons we keep them then in a circular ring and we used magnets to actually steer these uh steer these electrons to go around. And you can see um in the picture you can see max 4 again and in the big ring um the electrons go around a circumference of 527 m and they go around at almost the speed of light. And this is also quite important because if the electrons are bent or we make them go through a kind of slalom um they will start to lose energy and that energy is actually produced that light and that's the light that we then give to our users to study many different systems and we have applications from medicine to hard physics. So we produce light and we provide users with light and synretron light has very many very special properties. The the first one is that it's extremely bright. Um it's very very intense. If you look at a single ray of singleton light it's much brighter than the sun. Of course this we we talk here about one ray and the total output of the sun is of course much much bigger than a synretron but in that single single ray from a synretron the light is much brighter. Um, as I already pointed out, uh, synretron light has a very wide energy spectrum all the way from infrared to gamma. And it's tunable in the sense that we can through monochrometers and insertion devices pick out the light that we want. It's polarized um and that means that it can interact in different ways material and it comes out in small pulses which means that um we can even use a time resolved uh approach. I also should say that it's really really focused. So that means that it becomes a very very tiny ray of light that we can then match very tiny samples with. So if you look to a synretron um we have different different components that are being used to keep these electrons going around. Um so we always have an injection system. Um so the uh electrons are produced in what we call an electron gun and these electrons are then injected into the storage ring. We have then bending magnets that keep the electrons going around and these bending magnets are then also this one of the sources um where light is produced. We also have other insertion elements a wigler and an undilator and these actually create slalom kind of trajectories for the electrons and because there many ups and downs and the electrons start to wiggle they start to produce light light and it becomes stronger and stronger and stronger. So these really really enhance the light uh by many factors. um in the picture in the little movie that I now show this is from a synretron that is called Astred which is in Denmark in Orus. It's a very small synretron but the principles are the same. So we have the electrons that are produced in the electron gun then they're injected into the ring. Now in this synretron the electrons in the ring um are now bunched together so that they form like very dense little pulses where a lot of intensity from each pulse can be achieved. So now you start to see that the electrons are pulsed and then going through the banding magnets you can start to see that light is being produced. This light then goes into the beam lines that you can see um shown here. Um and then in the beam line they will hit a monochromator where the the colors where the color is selected in this case green. It falls on the sample and then in the detector uh the signal is then collected and the data is written um to the computer systems. Maxuru is a is quite a big singleton compared with with with um with um Astred which is really small but we work in the same way. Um in the top left corner we have our electron gun or our electron uh gun facility. The electrons go into a purple device which is called a lin. I can actually yeah a linear accelerator and in this linear accelerator the the the electrons are then accelerated to almost the speed of light. Max 4 actually has two different storage rings. Uh a lower energy one and a higher energy one. So depending on the energy uh if you want to have the lower energy the electrons are then pumped into the small ring or they continue get accelerated even more and then pumped into the big ring. Uh and then at both of these rings we have um the different beam lines. Now if you wonder how it looks like inside a storage ring um sorry this is a picture of our big ring and at the right hand side you can actually see a tube and that is the tube through which the electrons go around. All the rest that you see are magnetic devices. It's a lot of analyzers. It's pumping. It's water cooling. It's a lot of other stuff needed to uh to run our synretron. Now if we go to the beam line, um the beam line also has a typical build. Um so this is a very typical crystalallography beam line. U crystalallography beam lines are quite often almost the simplest beam lines that you can have. Um, and the elements, the big elements that they use are a monochrometer to pick out the exact wavelength. And then we use focusing mirrors to focus the beam. So to really make it the size of our sample, but also to place it exactly on the sample position in our experimental hutch. Then of course we have diagnostics because we need to know where the beam is and what shape and what intensity and to to really shape the beam we use slits. And then at the very end we have experimental setup which is in the case of a crystalallography beam line a defractometer which is actually uh a device that that holds the sample but also rotates it. Um we have a sample changer and a detector. And similar as for the um synretron itself, this is a picture of an experimental hutch, in this case Biomax. Um and what you can see is uh on the right hand side we have the sample changer. Um this sample changer can actually hold 400 samples. Um then in the middle where it says Arinox that is our very fancy sample holder where we can very very precisely rotate, translate and manipulate the position of the crystals. And then on the left hand side you have the detector. Uh and you look actually at the back of the detector. As a set we currently have 16 beam lines at max 4. Um, I put here plus one because we have a 17th beam line now under construction. Um, the hutch has been starting to build since last week. So, it's it's real now kind of. And these beam lines, they all kind of specialized for for their technique. Um, and they can be used for many different types of studies. And like I already said, um, one of the big things is that we want to make movies or we want to study material under what we call more realistic conditions. So that can be um looking at pressures, looking at heating up um looking at catalysis for a protein crystal um and and those kind of studies. And this is what we call INC2 inner porando. The other thing that we really want to that we really really um working on is multimodal methods. So combining for example defraction with spectroscopic methods at the same time on the same sample uh so that we get complimentary information on the same sample and I show actually um in my lecture later on uh why this can be important. So now I actually will go much more into the science. This was a short introduction of of synretrons. Um and I will go much more into the science and like I already said the three big methods that we are using are scattering and defraction, imaging and spectroscopy and quickly these actually show atomic structure, micro nanoructures and electronic structure. And all the examples on this slide are actually life science based. Um and this shows that that um synretatron has much to offer for life science including for work within viology. For us, um, one way of of working, um, with the beam lines and also with, um, associated laboratories and instruments is that we really like to think about problems in biology need to be addressed at different length scales. And um, we have a set of beam lines um that really allows to go to different length scales. So we have beam lines that are dedicated to to um protein crystalallography that's biomax and micromax where you then can look at the atomic length scale. We have beam lines dedicated to scattering cosax and formax um which really goes much more into micro structures and molecular complexes. We can go to imaging microscopy beam lines like nanomax and softax where we go from organels and cell biology kind of questions and we can even go to larger length scales using the tomography capabilities of DMAX and uh formax. We also have a a couple of associated or well associated instruments. We have the Lund University KUM facility uh for life sciences and we also have AFM infrared and optical infrared capabilities and these are highly complementaryary to to the beam lines and uh that we have. So I will now go into how synretrons have contributed for f viology. And the the way that I'm going to approach this is that I'm going to go basically from the right hand side of the picture to the leftand side and show examples on the way. There is one little excursion into infrared because infrared is very difficult to actually uh pinpoint in these length scales. Um but my main journey will be from left from right to left. So I will start with protein crystalallography and I think also historically um that's the right way to do it. So I will start with with a bit of um yeah history. Well, first of all, I I do think that it's probably the best known method within life sciences that is being used at the synretron. So, I cannot um not talk about it. I'm also myself a protein crystalallographer. So, um there is that as well. Um so we all know that for protein crystalallography it's important to really have a pure enzyme protein uh whatever you want to study or a whole virus. Um we then have to crystallize it and then we can collect data process the data and get the structures and get the hypothesis and ideas and uh and so on and so forth. So the big thing here is of course that that this method works with crystals. Um but it has been extremely successful and it also has been really important for phology and the first structures of plant and human viruses um of capsit of whole caps um they were already determined at the end of the 1980s uh middle 1980s and I think it's also noteworthy to to actually um observe that the first virus crystals were already obtained in 1935 by Stanley and this was some tobac tobacco mosaic virus and he didn't only grow virus um crystals but he also could show that these virus crystals were infectious which means that the particles inside these crystals were fully functioning and I think this is really important so the particles were fully functional know inside the crystals. [snorts] But um going back to these first structures of these plant and human viruses, um virus crystals, they defract just like normal protein crystals, but they defract weekly and the spots are very close together because these are huge complexes. And this means that you have to have a very clean beam and a very intense beam to actually be able to collect data. And here is where the synretrons come in. So already from from those structures um these were collected from from a synretron called chess in northern New York state in Cornell. um and it it it would not have been possible without without access to these uh to the synretron. So from the very beginning synretrons were very important. um more modern there have been really really huge capsites uh solved by crystalallography and and I think the record um that I could find anyways is blue tongue virus um by um uh Jonathan and now I just my mind is blank but it will come back because Jonathan Grimes he is actually a keynote later later I'll talk about that. Um but anyways um so huge virus capsets could be solved by crystalallography but I also should say um that nowadays if somebody would ask me oh I have I want to study a capset what should I do I would actually say use cym so I I just go a little bit in cym because I think it's really important to to say this Um, Cayama has well electron microscopy has of course been an essential method for fagology from from the minute it became available. Actually, one of the first electron microraph microraphs ever made was of a a mouse virus. That's the little picture on the on the right on the top. But for a long time, Caillou was not really the method to go into atomic details. and um this hampered the field. But since 2010 there have been really revolutionary improvements and now CAM is really the main method to study um virus capsets and also large complexes and the reasons are are manifold. Uh first of all Kyom is very good for large molecules. There is no crystallization required. Um you can work in the near native hydrated state by by um very quickly freezing your samples in glass-like ice. Um you can actually um look at many different confirmers at the same time. Uh which gives you really insight in for example distinct structural states like empty capsit or fully fully genome fil capsit and so on. And for the computational aspect, the fact that viruses are so highly um symmetric, this can be exploited in the calculations to really get very very high resolution structures. So cryom if you really want to study via capsit is the method to go to. Uh you don't have to use a synretron for that. Um I think you're much better off with with cyang. And you also can see this from um from the database at Fifer DB which is actually a a database that contains data of all capsit solved. And in dark blue on on in the right hand graph you can see the structures done by crystalallography while in lighter blue the structures by KEM are shown. And you can really see that until around 2010 this change came and and really since 2016 I think there are still a handful of of uh capsit solved by crystalallography but the majority is really by cryoam and that that's really logical. So I I think this is just a logical uh way that the field has de developed and and uh it's it's it's very good because you can also see that we have many more structures now and there are many things many insights that we have gained through looking at capsit uh structures. uh there has been a real understanding how how actually a virus can build up these very complex capsits with actually very few proteins. Um there is an understanding how how the capsit can be on the one hand very solid outside the cell but then once it starts to insert it falls apart and how it can then fall apart disassembly but also assembly again. The capsit have also given us ideas about where there are vulnerabilities um uh which then can be exploited either through vaccine or drug drug design. Um they also given us ideas for nanotechnology um make virus like particles to deliver drugs or genes or or or vaccines. Um and looking at the capsit from different families of of viruses, uh there are uh ideas about the evolution and the um those kind of relationships between viruses but even adaptations in their host cells. So I think the the studies of capsits have have been very very important but not only capsit proteins uh capsits have been studied of course by crystalallography. Um and again I start with a little bit of history um because um and these these are really beautiful structures of of um the spike proteins from influenza virus hemoglutin solved in 1980 and neurominadase in 1983. Um an extremely important pair of proteins. uh and here the structure of course have has gave us a lot of insight in how influenza adheres to the human cells. Um the scolidase activity of nurin days but also how actually the escape and antigenic drift mechanisms of the virus work on a structural level. Um neuromminades um is also from another aspect an important protein because it was actually one of the very very first proteins where rational drug design was used. So in the case of of um neurominades um the researchers um [snorts] Peter Coleman and his colleagues they really looked at the scaleic acid binding site and they observed that there is a little pocket which wasn't really explored. Uh it's a it's um pocket dominated by negative charged residues and they actually designed from from there um the first drug Rela. Um, Valencia is still on the market but it has also been modified itself further to to tummy flu and I think especially when the um I can't the was it pic I can't remember the name of the influence in 2009 but but tummy flu was one of the important drugs on the market um that if you take it just when influenza starts when the symptoms come for the first time it can actually really really lower mortality and um shorten the disease. So um this was really a success for for rational drug discovery. But of course many more virus proteins have been studied um and all these structures have given insight in many processes um from understanding and antion and recognition replication maturation and much more. And it is estimated and and this is just a very rough estimate that nine to 10% of the PDB entries are on viral proteins. Of course, many of them are copies of each other, but it's still uh an indication that this is really really studied. And um when corona was going on of course a lot of efforts uh have been done as well in in structure determination and already in 2020 >> [clears throat] >> uh most of the uh proteins in corona structures were available. So um for really understanding uh viruses protein structures are are a really beautiful tool to to to understand um this and synretrons of course play an important role in obtaining these structures and it's not only understanding the virus but also um obtaining insights in how the human body works um how our immune system recognizes virus but also how virus viruses can escape our immune system. Um and and um by studying different imunog globulins against viruses in detail, you you can get ideas that can actually also be used in vaccine um design. And finally of course and this is another example of a corona virus not SARS but another corona virus um working um in in complex with a protease which is important for the maturation of this this um virus and and here also by really studying um the interactions that the corona spike protein makes with this protease um it it creates ideas and it is important to understand um [snorts] how these ho how this process of adhesion but later also uh maturation occurs. Um protein structures also have played an really important role when it comes to antiviral drug discovery and I already pointed out the case for influenza. Um I think the other case really to point out is HIV because without without structural biology we wouldn't have many of these um inhibitors and it's in particular the inhibitors against the proteas against the reverse transcriptase and against the integrase and in the case of HIV um because of escape mechanisms of this virus and and the very leaky uh transcription system it has. Um it it really has been important to make cocktails of different inhibitors and we really needed the overall structural information to to get there. Um at Max 4 we also be working with uh drug discovery and um what we are using is fragment based uh lead discovery. So the idea is that we instead of big molecules like this HTS, we actually use smaller molecules to get an idea where things can bind and then by elaboration make a much quicker journey intowards lead that can leads that can lead to drugs. Now, one of the things with doing this kind of work is that this means screening of many many different lians. And if you have just one liant, it's it's actually not that hard. Um, you you soak one crystal, you mount it, you collect one data set, and you analyze it. It becomes hard when you have many. And we can actually collect easily 400 of these in a day and then it becomes problematic. And what we are doing is trying to make this easier. So we have um developed um together with actually LP3 and um Astroenica and Ceromics a facility for crystalallographic fragment and compound screening. And it it it um is built onto three pillars. Crystal preparation so that we can easily work with so many crystals. The data collection which is very um reliant on the stability of our high throughput uh beamline biomax and then tools to to really make the data processing and analysis much more easy to get into. Now a couple of um examples of the work. Uh so the first one is is again um is the results of um of work on um on a complex of two corona nonstructural proteins NSP 10 and NSP14 um where there were actually two crystal forms. um 143 fragments were screened. Uh and we then uh I mean um the researchers here uh which contained then people from LP3 uh as well as uh Max 4 and others um they could get 14 fragment hits. And I think one one of the nice things here is that it really also pointed out where the interfaces are. And it it gave not just information about what is druggable, but it also gave information about interfaces important for other interactions. And it's really the the the start of building a structure activity relationship for these proteins on which you can build more um on which you can then build towards proper leads for drug discovery. Another project um and that is going on is a is a project together with silage lab in Stockholm uh where we once again work with viruses. In this case it's um flavio viruses and one of the things here is actually of course it's it's interesting to work on the virus but it's mainly really used to get all the systems in place so that it becomes very transparent for the user uh to come and do these kind of experiments and this is in collaboration with CDCs which is a chemical biology consortium where you can do virtual screening of a vast chemical case together with experimental HTS um high high throughput screening and [snorts] together with us we're trying to really make this into a system that can be used for drug discovery for viruses uh for example so that's all I'm going to say about protein crystalallography and I'm now going to change a little bit in length scale and I go up towards small angle X-ray scattering Now small angle X-ray scattering is a solution method and um one of the things well it it's it it is actually in the nature quite simple in a way because what you do is you put your sample for example a liquid in a capillary into the beam and it will scatter and then you will will actually um collect that scattering pattern. Uh the scattering pattern is of course round but we then are smoothly integrated. Uh which means that we we we take circles and we integrate the whole circle. So you get a line going from the middle to the to the outside and you get then these kinds of graphs. And what you can see in these kind of graphs is that these graphs are really really dependent on the shape of the molecule in the in the solution. So if you have a solid disc, you get this this red um bumpy bumpy uh line. But if you have a long rod, it becomes this green, you know, almost straight slightly going down. So this sensitivity to shape and there is also a sensitivity to size uh makes that this can be really used to study u molecules. So it's a method to analyze the size and shape of of all kind of things. Um it it can be proteins but it can also be vesicles or it can be a combination thereof. [snorts] And um it actually covers a very broad broad range of sizes from 1 nanometer to 1 micrometer. And important uh to note is that the samples do not need to be crystalline. Um so it can be measured in solution and you can measure it under different conditions. You can vary the temperature, you can vary the pH, you can vary flow, you can vary a lot of different things. salt concentrations and so on. And like I said, it's sensitive for size. It's sensitive for confirmational change and also structural fluctuation. So what can you do with sucks? Um so you can look at protein size and shape. You can determine whether proteins are folded, unfolded or disordered. You can look at the oric state. Is it a monomer da tetma and so on. You can look at assembly and disassembly and I will show some examples. Um if you go to lipids you you can look at billayers and membrane structure. You can look at internal messes. We can look at higher organization such as lipid nano bodies. Um and we can actually look at changes in organization because we can actually study the effects of temperature, salt, pH and so on. And um we can do this in a high throughput manner which can actually mean that you you really look at formulation conditions. We can study protein lipid or RNA lipid interactions and we can combine it quite easily with separation techniques such as SAK or AF4. [snorts] So just a couple of of examples uh how people have been using um sucks in biology. So um both of these examples have to do with with with assembly and um maturation. And if you look in the in the left hand side, you can really really see that there is a difference at the bottom of this graph and the top uh between how these sucks curves look like. So the bottom lines are always for monomeic capset proteins and they don't they they don't really show features. But when you have a full virus as the top, you start to see these bumps. And in this case, people have now measured different sax curves uh in different concentration of salt and at different temperatures. And then by doing that they can actually start to think about assembly processes and kinetics of assembly processes. In the left hand side, it's a maturation of a of a capset going from a pro capsit into intermediate forms and to the real capset which is a kind of closing locking down um process. And you can see that that in this nicely rainbow colored graph that you also there can follow the process by doing sacks. So you can do these time resolved or or yeah series of sex measurements to to really understand um what's happening and uh chef at all in 2020 actually could by using these methods really start to look into the kinetics of the uh assembly and disassembly of a hepatitis B virus capsit where the assembly is much faster than the disassembly. Uh one is minutes and the other is hours and this kind of of work can was completely done based on on sacks and uh a bit of kio to to validate. we sax also has been very very important um for especially the way that nucleic acid vaccines have been developed. Now we all know that these nucleic acid vaccines the mRNA vaccines have been extremely um powerful a Nobel Prize probably the end of the corona pandemic um quite a lot um of of things happening there um and sex has been quite important to to really um getting there and I will actually FL Y. So now one thing about mRNA and and everybody who who has worked in a in a microbiology or biochemistry lab knows that mRNA is pretty easy to to fall apart. So it really needs to be protected and to protect it you know you use something which is called a lipid nanob body and um these are kioam pictures um of lipid nanobodies which is basically um uh it's it's I mean it looks like membranes on top of membranes on top of membranes so it's this very structured kind of lipid lipid monollayers that form these kind of complex nanobodies and these nanobodies they are really really excellent to give protection to mRNA but that protection can't be too good because they have to fall apart when they come into the body so the nano the lipid nanobodies they actually can easily transform membranes and then within the cell they have to fall apart because then the RNA has to be used to make the proteins. [snorts] So um the lip lipid nanobodies they have been studied by sacs really to see how different lipids and how different processes to produce them affect these uh these these nanobodies structure stability and performance and sax is really a good method to do that because it's in solution it works on these scales it's quite sensitive for for this kind of organization So, it's it's it's a really good method to do that. And we can actually because a sex measurement is pretty quick. Uh, a single sex measurements is probably not even a second. So, you you can easily collect many of them very very fast. So you can actually do systematic scanning of conditions and thereby really find the the um the the the kind of formulation the kind of set of conditions to make the thing that works for your particular vaccine. Even more, you can couple it to new separation methods such as um asymmetric flow um which makes it really an essential tool. So um this is another example from actually the cosax beam line at max 4 where we have an integrated AF4 system now at the beam line and people use this to actually look at um empty and cargo loaded liquid nom particles. So, AF4 is is really a nice separation method if you want to um separate small from very big things because it it is actually um it it tears it apart a little bit better than than the classical systems. Um and like I said um it's really really good for bigger things uh such as nanob lipid nanobodies. So it has been very important for vaccine development and um it probably um we will see also things in gene therapies and other um approaches. So now I will actually go into my very little excursion into spectroscopy. Um when I was looking through the literature uh of examples for uh on virus research um even though spectroscopy has been used in life sciences a lot um it hasn't been used a lot for virus work and I think one of the reasons is that um [snorts] most of spectroscopy works with natural occurring metals and there aren't that many inology. ology. Um so I think there are one or two examples but they were a bit ex es exoteric and I thought well maybe maybe not but vibrational spectroscopy um infrared and ramen and I will only talk about infrared are actually more commonly used and they also are available at synretrons um not at max 4 but for example at the sole uh Synretron and at um Solaris in Poland um there are u beam lines that are dedicated to infrared. So infrared is actually a really nice method um to look at changes. Um so what you get is a label free insight in in kinetics of cellular processes. And if you look at the different classes of molecules that are in a cell, lipids, proteins, nucle acids and and sugars, um each of them will give a a specific fingerprint. It will absorb at specific wavelengths in the infrared spectrum. And what you then can do is you can for example look at a cell not infected and an infected cell and then see if there are differences in these fingerprints. So it has been used a lot for detection of viruses uh even with laboratory equipment because infrared is also a very useful laboratory. Um but it can also really to be used to monitor cellular interactions and uh replication. Um and one example of such um um and there is I see a mistake in this slide which bugs me already but anyway I'll come to that. Um one example is is infrared of um hplex virus uh and what happens with increasing virus particles in the cells. And if you very carefully would look at all these graphs, you would see that infrared shows that there is an increase in nucleic acids when there is an increase of of uh virus copies. there is a decrease in the lipids and there is no change in protein and um it is um hypothesized that the increase in nucleic acid is due to increased number of chromosomes and not an HPF but an HSF sorry for that um and and um not only is there an increase in chromosomes but there is also an increase in damaged DNA due to due the activity of the virus. So you can follow these kind of cellular processes using infrared. There are also really nice examples on influenza virus um that that actually produces a um a co- protein an alternative reading of its genetic material produces a small protein that can form a kind of pryan or fibrals. And this also has been studied extensively by Inflet uh and I think we will see more examples in the years to come because it's an upcoming method. Then finally I will go to imaging and I will actually highlight um one method a bit longer than the other one. Uh and that method is actually soft X-ray cryotomography. And I should say Max 4 doesn't have this kind of beam line. Uh but it is available at other synretatrons such as um Hazard Bayi in Berlin, Alba in Barcelona and Diamond in Ditkot. And what the method does is that you have a cell culture that you then place on top of a grid. You freeze it. You plunge it in um in liquid nitrogen. So you freeze it very very quickly so that you get this glass like ice, you check it and then you you you measure it um with the X-rays. Um and you rotate your sample so that you get different projections that you then combine using computers to get a three-dimensional picture of the thing that you measure. Now this method um it works in the lower X-ray energies. So it's a low energy method um in an area where oxygen doesn't absorb the X-rays very well but carbon does. And that means that water which is oxygen it doesn't show up but all carbon based material will show up. So organels and membranes and all these kind of features of a cell will show up in in the um in the images. Um it means that you don't need to label. And the other thing I mean if you look at the resolution and that's actually shown in the in the in the slide to the left uh right sorry right um it's slightly lower resolution than uh cryop um electron microscopy imaging but the big difference is even though it's lower lower resolution you don't need to fip you can use the sample as a whole. So you don't have to reconstruct by putting all the layers back on top of each other. No, your sample stays intact. So it becomes um it becomes less invasive. Um and um what you can look what you can see with it is particle distribution uh particle um aggregation. you can see changes in membranes uh cellular architecture and all these kind of things [snorts] and like I say it's available at three different European synretrons and uh also in the US in San Francisco at the advanced light source and um also in Shanghai um at the Chinese synretron there and the kind of things you can see is is these kind of of pictures uh on the left hand side we we see infected lung cells with with uh SAS co virus uh where you then really can look at the changes in the different um in the different organals and uh the different membranes in the in the right hand side um there is um a case um once again have a simplex where you can see that these particles which are called NP which this nuclear p per p uh something. Yeah. The nuclear membrane starts to remodel and you get these small nodules forming and this method really could look into that how that works and um and and get more information uh in these cellular processes, how the virus actually um um modifies the cell and and how that works. Um you can combine these spectroscopic methods um with with you can combine this imaging with spectroscopic methods in this case fluorescent where you have virus um linked with green fluorescent um protein uh and you have um the uh endosomes in red fluorescent And combined you can start to to to see the overall architecture by soft X-ray tomography. And then you combine it with these other methods to really get a feeling on how these in this particular case a rio virus how it tracks into the cytoplasm and actually goes into the endoome and uh is is encapsulated there. Um finally I would like to to say some words on heart x-ray tomography. This is really an upcoming method. Uh and it it really can be used for looking at tissue and it's it's somehow related to what one does in a hospital. Of course, in a hospital, the patient lies still and you have the X-ray source circling around and you get the the pictures. We can't do that in a synretron. Instead, we have the sample uh that rotates, but the principles are the same. And this method really uh made a lot of breakthrough also during corona. Um when looking at tissues uh from patients or from some actually pathology um samples uh people could really really start to see the damage at a very cellular level that corona had caused. Uh so in the heart there was definite vascular remodeling uh happening and also in the lungs you start to see very abnormal um patterns in in in the blood vessels. Uh and um this was beautiful work and and really the first kind of structures of this level and I think uh there is a lot more to come um in this area. I also want to point out that I'm not the only person that thinks that these methods are really important. Um, and I think this is an extremely um um how do you say um yeah, it's it's a very nice project to follow and it's called Project Orion. It's at um it's in Brazil and it's actually the the um construction and building of the of a PSL4 lab um the first in Latin America. But what is special about this particular project is that it's actually linked to the serious um synretron in Brazil and Sirius is very very similar to Max 4. So it also uses MBA technology. It's only a couple years older and maybe one or two years older than Max 4. So it's very very similar. And they're going to have within this BSL4 laboratory um three beam lines that are part of the project. Um CB Pirina which is a soft X-ray tomography beam line as I just showed. Timbo which is a tender. So tender is in between soft and hard. This is actually quite unique because the arnot I think this actually the first of its kind. So we will have to see what it will produce. And then hibisc um which is a hard x-ray tomography beam line as as shown in the last example and these all will have bsl4 capabilities somehow. So I think this is extremely ambitious and it will um be really really nice to follow what actually comes out of this. And um this also shows that that there is still a lot to be discovered because some of these methods they only are 10 years old and they were mainly used by pioneers but of course it has to become common track now. So the last um five minutes probably of my talk I will talk about um how to actually get beam time because I think um if you have ideas um especially um for saxs for example but also we have infrared in our hub also in our facility not at a beam line but as a site laboratory. um if you have ideas uh then then I think it's good to talk about this as well. So any experiment at at the synretron is actually it builds out of different blocks. First of all um you have to have of course your scientific question. Then you have to prepare your sample and your experiment. You do your experiment. Then you have your data processing and then finally you get your results and um so what we offer at max is of course um the experimental time but we also have additional infrastructures for sample preparation as well as computing and I should say that for sample preparation we are actually um adapting the lab at the moment to that BSL2 u capability. so that we are actually able to to handle level two um samples. Um I think it's important to say that we not only offer light or x-rays uh and instruments to our users but on the technology side we actually have a lot of expertise in our building. um people who know a lot about what is possible and what is not possible and that is also part of the offer to our users and we do have a data storage and analysis analysis cluster um it doesn't go the whole way but it will give a really start good starting um position. So there are two different ways to get beam time at Max 4. The first one is through peer review. Um this means that if you have a really good idea, you you compete uh with other researchers. Uh through an open call. This is then um gone to a peer review. uh proposals are then ranked on scientific merit and then if you're if you're high then you will get beam time and that beam time is for free. So you don't have to pay for it. What has to be done is that if you get results they have to be published. If you don't publish and you ask for more time at a certain time it will be no you will not get more time. Um you can go the other way. um which is if you can't publish because it's proprietary and there are there are things that you don't want to publish then you can pay for your beam time and it goes through a contract. Um but this is really uh mainly used by industry. We actually don't allow academics to go through this route. We don't allow academics to pay for beam time. they have to be reviewed in competition. We do however have a system which we call fast access and I think fast access is actually a really good way to test the waters to test whether things are possible. So we offer different uh fast access modes. Um the first one um is a mode for scientific urgency. That can be a PhD student that needs to finish a last measurement for a paper so that the thesis can be produced. Uh it can be that you sit on a sample where you know that there's a lot of competition. Um those go under scientific urgency. Um you have to make your case of course. Um we also on some beam lines offer standard measurement that is a very precisely described measurements that we do. Um no no extras but it can be really important to get that one spectra that you need for a paper. But then we have something which is called feasibility. And this is really a short experiment where we look if your sample is feasible. Do we get anything? Do we get any signal out of it? uh but also would actually the data that comes out of it help to answer your specific scientific question because quite often if you don't know the method and we don't know the science it's it's not always easy to know that the data actually will really help. Um but feasibility uh should actually lead to a general proposal that then would lead to full beam time. But it is very good method to come in and and and really get a feel for what it is to to use a synretron. So if you have an idea of an experiment um what you need to do is to contact us uh to discuss what beamline would be the most suitable and then really really important is contact the beamline staff and discuss your idea. So once you know what beam line is the most suitable really contact the staff because they can really help. [snorts] They can make um the design of your experiment such that it is possible and then of course you you have to also decide what kind of access you want to do. Um I I will not stop here. Um I think somehow we need to have this on a labs web page but contact people for life science for the different beam lines are in this list. Um we probably will produce this on our website one day in the not so distant future. And um with that said I I'm almost finished. I just want to actually highlight two things. Um this was the first Panda webinar but there will be more. Um the the next one is already um scheduled on the 13th of August on 50 years of Ebola viruses by Nadine Benedov. And then on the 26th of August we will have um a webinar on protein production by Marlon Beex. And then finally we will have in um in be the beginning of November our panda symposium um which which goes much more in detail on many of the things that I'm talked about. Uh we will have keynote speakers Jonathan Grimes famous by the blue tongue virus that I showed earlier but he will actually talk about influenza uh Mel Landau from DIY um on on antimicrobial and peptide amoids and then finally uh Marian Janas Arteta uh who actually is going to talk about these lipid nonobies and mRNA And then my final final slide. Um, if you're interested in Panda, use the uh Q code and uh you get much more information. And I would like to thank my colleagues at Max for for um some of the slides and thank you for your attention.