So for example, this is a 2004 nature paper by Brunger's lab, okay? So what they did is, they used the protease, okay, and then they can crystallize the protease is substrate. So in this case, the protease for the Botox, there are seven different kinds. So this is Botox A. And previously we talked about Botox B. So they recognize all essential machinery for transmitter radius. For this one, this Botox A, recognize so-called synapse 25 and other important proteins. Okay, so by crystallizing it. Okay, so this is the crystal structure. Again, this linear stream indicates the substrate, synapse 25. And this square-like structure indicates that this is the protease, okay? And then they can crystallize it, and then they identify, this is how to the protease, which sees inside, can recognize a substrate, okay? But then you will ask, as you just mentioned, the protease will always cut the substrates. How can they achieve the high affinity? Because of crystallizing. In these two we have the high affinity together. Indeed for this paper, we also have to engineer the protease, and the substrate to mutate that catalytic activity only allowed the protease to bind to substrate, okay, without cutting it. Introduce two mutations, for example. So then they can capture this high affinity state that the substrates bind to the protease, but without cleavage. Okay, and from this structure, actually it is a very unique principle, how the protease, the Botox and toxin achieve this high specificity. Because, as you can see here, the cutting site that you will recognize this substrate, bind to the protease actually using additional substrate structure in other sites to bind to the substrate. Okay, so rather than just recognize a short peptide that had been cut, this protease can recognize a whole list highlighted here. This part they will interact with the synapse 25 before cutting. So this is, for instance, why this protease is so specific. Okay, so it only cut is substrate, because if it's a short peptide sequence, you expect that as long as it happened in some protein sequence, you will be recognized. But actually it's not, you recognize a very, very long domain using this protease 3D structure. Okay? So this is illustrates after identification of the substrate and protease interaction, and people using a structure status to further achieve at atomic resolution how the interaction occurred and how the specificity is achieved. Usually it's not in the other direction around. That is, usually people do not crystallize the and then they can guess what might be the substrate. That might be too difficult, okay? Only upon understanding their interactions people can fully optimize, and at a higher resolution trying to understand their interaction. Okay, so this is one example illustrate how the toxin achieve a high specificity to recognize a substrate using a slightly different toxin, but at the same principle. And how does the toxin get into the cell? Get into the membrane and cleave its target? Because all the substrates are inside of nerve terminals, inside of cells in the cytoplasm. But to the toxin, usually happen, provided by the bacteria and this is how it is being achieved. First, people identified what is the receptor that mediate the internalization or recognition of this substrate. So, previously, we already know that this toxin are composed of two parts. One is the light chain, the other one is the heavy chain. And the heavy chain is important for recognize the binding side. So, again through gassing and screening, how do they screening? They can screen only the protein localized in the okay? So people actually found that synaptotagmin, SYT. It's locate in the plasma membrane and it can be recognized by BoNT/B heavy chain, okay? So if you look at the whole toxin, the toxin can be composed of the light chain and the heavy chain. So the heavy chain that it specifically recognizes one of the protein components, synaptotagmin, which happen to be the calcium sensor to sense transmitter release. And there's another lipid modification that this protein recognize, okay? So indeed, this protein using the heavy chain to recognize two receptors simultaneously to achieve high affinity. And people believe recognition of that receptor and the fuller internalization, the lower pH inner vesicle will lead to this light chain translocate through to the protein port that are formed by this heavy chain. So now light chain will get deliver into the cytoplasm to cleave its target, okay? So in a cartoon, this is how, The bacteria toxin works. Okay, indeed it's a very neat mechanism. In a nerve terminal you have synaptic vesicle and with a lot of different proteins associated with it, okay? During the vesicle fusion, The synaptic vesicle protein there are lumen side in this case. Synaptotagmin 1, this lumen site actually will be recognized by the toxin heavy chain. Okay? And with the lipid modification here, okay? So how this toxin really works? If all the vesicle are not fused, then this synaptotagmin will never have been reached to the plasma membrane to be recognize. They're just hiding inside the vesicles, okay? But how this toxin works is building a transmitter release. Then this synaptotagmin will get its post to the surface. And the heavy chain will recognize it and then binds to it. And once it binds to it, during the internalization, this vesicle gets further internalized. It already has the synaptotagmin 1 here and it has this heavy chain. And this a light chain, and that's a lipid modification here, okay? And because of this is light chain, okay, because of inside vesicle, in that case is lower pH, the proton concentration increase. And people find out actually, doing this the pH changes. This heavy chain will insert into the plasma membrane to form a pore. A channel that will allow this light chain translocate into the cytoplasm. And the light chain and heavy chain, their interaction is through, by. So once they're into cytoplasm then this light chain, it's a protease. You will immediately recognize a lot of essential synaptic protein. For example, another protein which is synaptobrevin which is localized just at the vesicle surface, okay? So this light chain will just be cleaved. And since this synaptobrevin is so important for transmitter radius, there will be no transmitter radius, okay? So essentially, this toxin is a very smart toxin. The only target the active synapse, well, that's the transmitter release, okay? And once it came to a cell, cleave the synaptic wiring. There's no further transmitter release, okay. So the toxin will no longer target because of this this is synaptotagmin that's no longer exocytosis. Okay? So then this toxin will efficiently to target other active synapse. So you will no longer be translocate into it. So it's being very efficient into controlling or blocking the transmitter release. It has all these weapons, that one state verses the other to recognize the substrate, delivered the weapon, and the weapon will attack the host. And then the host exocytosis and you can save the energy to attack other ones, okay? This is pretty neat, right? This structure illustrates that how they recognize. And it turns out that this synaptotagmin actually also an essential protein. It can sense calcium, it has this c2a and c2b that can bind to calcium, okay. And actually that's how they sense calcium and trigger transmitter release. So evolution there is this neat process, okay?