Hello, everybody. This is Linda from Brain Education TV, and I'm David from Body and Brain. Yeah, David back for another live. Thank you. Nice to be here. Yes, it's always good to have David in our conversations that talk about more about brain education practices. So thank you for being here. My pleasure. Everything seems to be working. Yes. How's everybody today tuning in? I'm excited about talking about this topic. Yeah. Today is topic is something that's really important to our practice. So before we start, could you guys type in the chat, your name, where you're tuning in from? We always like to see where you guys are coming in from all across the world. Yeah, you have a global audience, which is good. Yes. All right. So those of you who are new to our YouTube live, I just wanted to let you know we have a super chat function. So if you're somebody who's a fan who's been watching some of our videos and you want to contribute, then please click the little rectangular box under the place where you chat with a currency symbol based on where you're located. If you click that, you can donate a small amount to our channel to help under contents. Any and all donations are very, very much received with gratitude. Yeah. Yes. All right. So let's get started. And yeah, while while we talk about this topic today, please feel free to type anything that you want, where you're tuning in from if you have any questions, comments or anything you'd like to say about the topic, then please do so at any time. Yeah, I wonder if people have heard about this topic before. I know, right? It's very, very new. So it's actually also very ancient. Right. Thousands of years old. To many people, maybe new. It's new to most people. Yeah. So what is that topic today, David? Today's topic is Hongik. Hongik. Oh, there's some unfamiliar words suddenly. Hongik. So Hongik, which comes from Korea. Yes. It's kind of a philosophy. It's a philosophy or it's a principle. And it most often refers to education. So what is it that Hongik means? Hongik means widely beneficial. So widely beneficial in terms of education, kind of education where the person who's learning is not just memorizing facts and developing their personal skills to be successful in their career only. They're supposed to be educated so that they can benefit the whole community. Like that's an inherent part of what they are learning. And I think that's really cool. Yes. So can we all repeat Hongik? Hongik. Hongik. You could write it down in Korean and show them the characters. Oh, okay. That's a good idea. Hongik. So traditionally, yeah, education, but there's a lot wider application of Hongik than just education in schools. It can also be like your culture is a Hongik culture. Your family is a Hongik family. Your government is a Hongik government. Everything can be Hongik because ultimately we all have to get along and work together. And that's what Hongik is about. So where does this idea come from, David? Where does this come from? Yeah. So for people who have never heard this term before, like is it what language is this Hongik? I believe this is Korean. You might know a little bit better than I do about that. But this is from Korea. And then many people say that this was the founding philosophy from ancient times. So ancient Korea that has been there for thousands of years. Yes. So Hongik basically, for those of you who are joining in Hongik means benefiting all life, widely benefiting all life. Yep, widely benefiting. So you can can be a Hongik person, a Hongik in gone. We often talk about that through our brain education, body and brain practice, that we're not just trying to become more flexible, or be able to clear our minds for ourselves only, but to become a Hongik person who benefits all. Right. So David mentioned that it comes from Korea. But the thing about it is, you know, I'm Korean, and I feel like modern day people in Korea don't really know about this concept. Yeah. Even though historically it comes from that area. And actually, the country of Korea was founded on this idea of creating a Hongik society, a Hongik, which means a society that works to benefit all life. However, as Korea has modernized and adopted other cultures and ideas, it has lost the real meaning of the founding of the country. So modern Korean people don't really even know about Hongik. So if you lived in Korea or if you just spoke Korean and you heard Hongik, what would you think of? People would have heard of the word, but they wouldn't truly know what it means. Okay. So there's a lot deeper meaning behind it. Right. Interesting. Which we will talk about today. Well, and then today's society, we have a lot of benefits from competition. I would say that there's a lot of competition built into our system. It's winners get more.  But something that I think of is brain education creator Il-chi Lee has often said in that system, there's always a lot more losers than winners. Is that really the system that's going to make us all the happiest and healthiest? Right. And in our success driven society, we oftentimes just, when we want to do good for the world, we think about sacrifice. We think about altruism, you know, giving to the community, giving mother Teresa. Yes. Giving to others unconditionally and just give, give, give, give, give. And I know many people try to live that kind of life. But if you're somebody who tries to give, give, give all the time, you will quickly realize that you get, you get drained and you start to feel a little bit of resentment because you start thinking, OK, I'm giving so much care and love to other people. But what about me? I hear that a lot. People who are natural givers, they always say, what about me? Who takes care of me? And it's just a natural feeling that arises when, you know, you just give, give, give, and you don't replenish yourself and you try to find somebody who will replenish that. But so naturally you look for someone else who's a giver. Right. And then it creates this cycle of like, OK, I'm a giver, so I'm going to give everything to other people. And then now I want somebody to give back to me, which is typically how we operate in our society now. But often that's really difficult because a lot of times I don't know if this is everybody, but how about you guys? If you're a giver, sometimes it's hard to receive. That's very true. Giving is much more comfortable, but actually sitting still and let someone care for you is less natural. Right. So what I really love about Honeak is it's very different from sacrifice that we're used to thinking about, especially in the West as a form of like model citizen, someone who sacrifices. The hero always sacrifices their good for the good of other people. But the clear difference between Honeak is like a fine line between Honeak and sacrifice is that remember in the chat, I also wrote, Honeak is widely benefiting all life. When we say all life, that includes my life. Because I'm part of the whole. I'm not separated from the whole. So you don't have to just kind of, you don't have to.  Right. And Honeak actually says you shouldn't be like that because that's not sustainable and it's not good for you or the other person.  Interesting. So I could see where there'd be some question like, OK, that's a great ideal. Ideally, yeah, I want to benefit me and everybody. But it's sometimes it feels like it's either me or them. Like, what can I do? What does it mean to actually be Honeak? Yeah, so. Yeah, we think about a lot of like separation between me and other people. And then also, I do want to mention that like a little bit more about sacrifice is that like, you know, when you guys, when you've ever been in a situation where you like give to somebody, of course, there's a good mind that goes with it. But underneath at the very like bottom, there's also some kind of expectation. Like because I gave you this much of my time and energy, I expect when I need it, I expect you to give me that kind of time and energy back. So there's this kind of like unspoken expectation and calculation because I gave you this much, I expect this much in return. And if that this much I want back in return is not given to me, then we start getting angry, resentful, lonely, sad. You know, that makes me think of another aspect of giving. I hear a lot of people say it's good karma. OK, I'll just do this good thing and it's good karma. I think that's really wonderful and true. But then this is something we're going to dig into a little. If I am not connected to myself, then the idea that the universe is just going to give something back to me, I think it's still also a little bit of separation. Right. And then so now at this point, you guys might be wondering, OK, so then is there a difference between Hongik actions versus non-Hongik actions? And again, that's another kind of way that our brains like this way. I'm Hongik. Yes, good. If I don't, I'm bad. Right. Like this is a list of Hongik actions and this is a list of sacrifice actions. Like it's not like that at all. Hongik can also mean a mindset of how you view a situation. So for example, let's say that I gave David something, you know, because I looked at I found something and I was like, you know what? I remember David told me that he really, really wanted this pen. And now I saw this pen and it was on sale. And I just really felt like if I give this to him, he would feel really, really happy. Maybe it's on sale because the clip is broken. I don't know if they can see. So let's say that I gave David something. And typically what we would expect is, OK, I gave David this. So I kind of expect that if David finds something that he will give me something back. However, Hongik, a Hongik way to look at this is I gave something to David and then I David reacted very, very joyfully to it. And he was so, so grateful and I could feel that it was a sincere sense of gratitude and a sincere sense of joy and I witnessed it. So then I could feel like even though he didn't give me anything material back, he gave me a certain energy and sincere gratitude and joy that that emotion itself is already gift enough for me. So in that I created this happiness and joy for this person, I already feel fulfilled because that made me feel good about myself and my actions. What happens when the people that you're trying to help and care for don't appreciate your work? Right. So because I think that happens a lot. That happens a lot, too. So then that gets a little bit trickier because then you have to bring the focus back on yourself. This person might not react the way that I expected. Yeah, I might be like, hey, this isn't a perfect pen. Right. That's what I wanted. That's his choice. He has the right to react however he wants. However, when I look at my actions with this kind of observer, observing my action and I see that my desire to help him and give him joy was very sincere than just recognizing myself for that. Even though he doesn't recognize it for me, when I look at my mind, when I look at my actions, I know that I did it with pure intention and I feel good knowing that I brought forth that mind and acted from that mind. That's interesting. So there's this sense that observing yourself is important. Yes. Wow.  Right. I'm impressed with myself. Well, I'm touched by my own sincerity. Often we can see somebody else do something and feel that. But it's really powerful. If you, in my experience, if you feel that about yourself, it's really heartwarming and actually healing. Right. And that really boosts your confidence and boost your confidence. Yeah. I think that's a big part of Hongik too. Right. Being able to watch yourself and say, yeah, this is what I want to do. This is who I want to be. Yeah. So when we go a little bit deeper into the Hongik, there's this idea that in our society, there's separation. And then we talk about oneness too. But, David, can you give a little bit of example about how we can view Hongik in terms of oneness rather than separation? Well, I think what you just said, when we do our best, there's always a limit to how much we're going to get back physically, financially, even emotionally. There's a limit. So as long as we feel like and believe that we are separate, it's really we're not we're not making a profit energetically. We're giving more than we're getting back because that's just the nature of the world. People don't recognize it right away. But inside, Hongik is based on this idea that inside we are all one. That that's not just the philosophy. That's a real thing that we are aware of that exists in our brain as a sense of oneness.  They're just, you know, the family or their deep friends. There's love there. There is a oneness, which is very natural. And in that case, doing something for others with no expectation unconditionally feels really good and fulfilling to me. I never need any emotional appreciation about it. That is a natural thing that I believe we all have inside and we can develop. But it takes time and it's natural not to feel that way all the time, like sometimes be disappointed not to get that emotional appreciation right back. But I believe we can practice it and get there. Right. And something that I also heard about Hongik that like really kind of made me rethink about how I treat people and view people is that we already know the sense of like oneness with each other. You know, that's a very like common idea and term that we, you know, talk about in society. But if you truly, truly know that we are all one, then when I hurt somebody, because we are one, I am actually hurting myself. And then when I, on the opposite side, when I do good for somebody, if you truly can feel that we are one, then doing good to you is doing good to me. Yeah. Yeah. Have you guys felt that? I mean, yeah, take the other example, like you mentioned. Oh, I, everybody's in a tough situation. Maybe it's a heat wave or maybe there's a drought or whatever. And I just got to look out for me. Oh, I'm safe. Oh, of course I feel relief when I'm safe, when other people are suffering. But am I really happy? Do I really have hope for life if I'm the only one who's safe and everybody else is suffering? I really think if we're sensitive, it's really clear we can't be happy that way. Right. And even if we suffer a little bit more by trying to help others, we are actually more fulfilled, even if we suffer a little more. Right.  I'm sorry for them. Yeah. I mean, like, I wish, I wish things were good. Sorry, guys. I'm not going to increase my suffering because then we both be suffering. And what would that be? Right. So that exactly itself is this idea of separation that is causing so much isolation and hurt and suffering and loneliness out in the world. Because now more so than ever, it's so funny because when we weren't as well off as a country or as a world, when we weren't as affluent as we are today or as comfortable in our society as we were today, we needed to help each other. Well, and that's a much more recent thing in Korean history as far as I know. American history, if we're talking very generally, it's been a few generations that have had a lot of wealth and technology. But in Korea, it's a much more recent experience of like having nothing and then a big change very quickly. Right. So yeah, this idea of like, if if you're suffering that has nothing to do with me, that's that separation mentality. That is the opposite of Kongik. Kongik is a higher consciousness that really, truly realizes not just as an idea, but in your body viscerally and in your life that because we are one, I cannot do hurt to you because not because it's bad to the other person, but not just morally, right? Not just morally, but hurting you actually hurts me because it doesn't make any sense. Yeah, right. Whatever I would get benefit, beneficially out of it is not worth it. But I think it's important to say that this is not just a personal development or morality system. In this day and age, this is really important because we live in a global village. We are not separate. Again, those illusions of separation are now breaking down even across different countries. If one country is experiencing an invasion and poverty and war, everybody is affected. And we could be cynical and say, oh, yeah, it affects our economy, but it doesn't really affect me. But think about it. If you're seeing news about that suffering and actual video from the ground and hearing those people's voices, don't you suffer? Aren't you connected to that? I know I am. Right. Don't you feel an emotional wave because of that? And want to do something. Yeah. So Hongik is not just, as I said, personally beneficial or my local community. But the problems we face as a society, we cannot solve those problems just with more technology or different government policies alone, although that could be important, or a little financial change. Like we need a change in consciousness. Right. So before we get into that, I just wanted to say hi to everyone who's been typing in the chat. Yeah, yeah. So hi, Judy, and core love. And then you, Myung Soo, hello from Korea. I think it's turning in from Korea. Ragey, Village Nature, Maria, Bill, Bumperville. Hello, everyone. Thank you so much for joining. Good to see you. Oh, I see a comment here. OK. The important thing is what we do. Pure motivation is the point, not attachment or expectation. I think so. I agree. There is an ethereal net that connects everything, promise, energy, we are energy. Yes, Maria, you're absolutely right. All your comments are absolutely true. So, you know, all, speaking of that, like all the saints and sages, yeah, kind of talking about this similar idea and concept, just in a different culture, during a different time, with slightly different words, but at the core of it all, it's all the same teaching. Yeah, basically, that we're all connected. Yeah, exactly. OK, so David, you talked a little bit about consciousness before we went on this little break. So we mentioned that Hongik benefits other people, and then it's not just my life, but as the world in general. Maybe some of our audience members, who this is their first time listening to Hongik, listening about Hongik, they may want to know, so this sounds great, but I want some concrete ways how adopting a Hongik lifestyle can really benefit me. And what that means for me. So I think that has is linked to consciousness. So can we talk a little bit about that? Yeah, do you think we should talk about individually or as a society first? I think maybe individual and then work our way up bigger. Sounds good. So individually, and I can speak a little bit from experience. OK, individually, Hongik, for me, is actually a way to have less stress and more happiness and more confidence. As somebody who is interested in volunteering and helping others and creating a society where more people can be healthy and happy, I feel a lot of stress if I'm not clear on how to help. You know, how about you? I mean, I want to do good, but sometimes I'm not sure what can be good. So when I learned about Hongik and both what is good for me and what is good for other people and how that's a practice, it really gave me more confidence to be active in life instead of trying to hold myself back. I'll give you an example. For the environment, I don't want to create more waste. I don't want to drive my car like do all these things. And at the same time, I want to live. I want to I want to experience things. So when I'm Hongik, I find, OK, it's not just about having the rule, like create zero waste, although that's a great thing. It's about what is my bigger activity? Am I helping people? As a society, can we reduce waste? As a society, can we care for each other? Because that's going to be more important about saving the earth and saving energy than anything I can do individually. So Hongik mindset actually gave me a lot less stress because I didn't have to judge every single thing I'm doing and say this determines whether I'm a good person or a bad person. So I think that's something that helped me a lot. So how does Hongik change consciousness? I really like this idea of changing consciousness because there's so many things that are happening and we're also trying to find answers to so many of life's problems and society's problems. And one of the things that Brain Education recently talks about and it'll cheer the founder talks about is this change in consciousness to help these societal problems. So now that it's going from the individual, let's work a little bit bigger. So how does Hongik mindset benefit a society? I think the first question that some people may have is what exactly are we talking about when we say consciousness? That's true. So... Right. What is that? Is that mine? Many, many, many different ways and implications of consciousness but one of the practical ways is my consciousness is kind of the fundamental information and identity. Who am I and what is important? How do I judge the world? How do I interpret the world? There's a fundamental kind of structure of the mind and information. It's like the lens that we look through and we can look at many different things with the same lens. Ultimately, there's not a lot of change but if we change the lens, we can suddenly see much farther, much closer. We can see a whole different world and often the solutions to our challenges are seen from changing consciousness, not just looking at a different information through the same consciousness. I don't know if that's really clear but consciousness is like the lens we look through and when we change that, we can see a whole different set of opportunities. So it's like kind of like the level of our mindset that we're operating from? One way of talking about consciousness is expanding. Expanding consciousness or brightening consciousness. If you're looking through a lens and the aperture is really small, there's not enough light, everything looks dark and you don't see any beauty, no possibility. Yeah and you only see this much. And you only see this much. You open it up, you expand it or you brighten it. You can see a lot more and you can do a lot more. I like that analogy. Yeah. So consciousness is important and it's a thing that we can develop. But it's not just understanding. It's not just conscious learning. That's the difference and that's why we do physical and energy work as well because it takes more than just learning a new piece of information to really change consciousness. So how does changing consciousness which is tied into this Hongik life set and mentality, how does changing consciousness and Hongik how does that affect any change in the world? In the world, you know, we're more and more aware of what our leaders are choosing each and every day. And I think one thing that becomes more apparent is that even leaders, a lot of them make choices based on very personal emotion. Yeah. Personal feeling. Personal gain. Personal desire. Money. Lobbying. And you can't say that that's like wrong. Nobody is, you know, perfectly immune to emotion. But the question is, how can we make better choices? How can our leaders make better choices? Benefit more people, not just their family. And then how can all of us, the population, ask for better choices and better results? One of the ways is expanding consciousness. Think about a child. Now we imagine that children are really pure and beautiful but they can also be really, really selfish. In fact, that's natural and healthy for them is to be really selfish when they're young. They need safety. They need energy. They need love. So they're very selfish about those things. But as we grow up, we don't need to be quite as selfish. We can trust. We can share. We can learn to make harmony. And that's a natural process of growth. And in a way, that's consciousness expansion. And even though we turn 18, it doesn't mean we're done growing. Our consciousness can keep growing and expanding throughout our lives until we're 80 years old, 100 years old, and beyond, we can keep growing. So a big thing that we talk about in brain education is changing consciousness, as David mentioned, changing consciousness as the root of solving life's problems because somebody who is operating from that higher mindset, from that wider lens that you're talking about, and seeing more things and acting and behaving in a way that benefits more people with a bigger mindset. If you're operating from that kind of energy, it's impossible to behave in a way that jeopardizes a certain race or puts a certain group of people down because you're seeing in such a bigger light. So it seems like our issues of social inequality, politics, poverty, disease, homelessness, environmental destruction, war, famine, all of these things come from people, like many, many people around the world out of the 7 billion operating from a small outlook on life and looking to only benefit a small group, maybe their own family or their own political party or their own something rather than the whole. Yeah, because they don't see that it's a possibility, not because they're evil. Right, right. Because they've only taught to look this much, maybe from their parents or even just, I feel like society in general, this success-driven society that we kind of are going back to from the beginning of this life, this success and competition-driven society from a very young age, we're taught to look only this much so that I succeed. My people benefit. I do better. Who cares about this other person? If I'm doing well, then this person, whatever. Or even if I care about them too bad, only one person can win or only one country can win. Bigger consciousness says, even though it's hard, we can achieve harmony together, peace together, health together. Right. So David mentioned at the beginning, or not at the beginning, but during this little segment that how his personal life changed from Hongik. So I wanted to give a short example of how my life also changed from Hongik. So as I mentioned, I'm Korean, but I've never heard of Hongik before brain education. I've never even heard of this word before, right? Because it's not something that modern Korean people talk about often. But when I first have heard about Hongik, I'm naturally somebody who gives because I'm the oldest of three kids. And so I was the big sister all the time. So from a young age, I had to yield. I had to share. I had to give. And I had to, you know, even in my friend group, like I'm the like take caretaker, the giver, the person who like calls everybody, brings everyone together kind of person. So yeah, and that made me feel good. It made me feel like I was helping people, but I would be lying if I said that I didn't ever feel like, what about me? You know, that's a thing that I also said very often. I'm taking care of all these people, but who's taking care of me? And then trying to find somebody who will take care of me too. So and that's the only kind of like service or care that I was taught by society. I didn't know how else to function besides give, give, give, give. And then eventually just hope and pray that somebody will give me too. Which is a tough state to be in. It's not good because then you're constantly seeking approval and attention from other people. Anxiety. Yeah, who wants that? No one wants to live that way. Nobody wants to stay there very long. Yeah. So when I heard about Hongik, this idea of widely benefiting all life, which includes the people I love that I'm doing this for already, but also including myself. Just hearing about that concept gave me comfort. Gave me this like, there's a way out for me to feel love too, to feel cared too. That there's an idea that goes beyond sacrifice that encompasses a wider group, a wider kind of perspective that I've been seeking for. So Hongik I really felt was the answer that I've been looking because it benefits the people that I already was loving and benefiting, but it also made me realize that I'm valuable too, that I'm a precious being too, which I had never really thought about that before all the people I loved were precious and they deserve love, but I never thought that I too was precious and I too deserve love. That was a very foreign idea to me. So important because we know that you can't really give that healing love unless you can have it inside of you. Right. And that's exactly what we're talking about is you've got to be fulfilled inside.  So things are possible that didn't seem possible before. I always think when we talk about war and poverty and famine and all these problems with the environment, it seems like, oh, they're too big, there's no solution. But if you look at it, there's actually a lot of good solutions out there with technology and science and certain governments doing different things. But we don't choose them. We don't focus on them because we're looking through that lens again. It's different. We don't even think it's a possibility. So if we want to change the world, we have the capability, I think, to make a lot better situation for many people and life on this planet. But consciousness is the key to actually seeing it and then doing it. And because Hongik elevates consciousness, this is why we titled this live, Hongik, A Korean Enlightenment Philosophy. Right. Enlightenment is not some separate thing. Enlightenment is all about oneness. So let me take some comments. So Maria, you are right. Evil grows when good people don't do that. That's true. Yeah.  Hongik, I totally resonate with that one. I was always giving so much love. I'm just realizing I'm better and I have to love myself. Yes. Right. Really important to practice that. Yeah. There are so many of us, give, give, give givers out there who forget that you are precious too. And then sometimes it looks like, why are the takers so successful? Why are the takers that people who look like they're only concerned with themselves, why are they in power? But it's up to those of us who want something different to also have power. And that's where Hongik means connecting to myself. If your mind and awareness are outside yourself, there's no way to gather that power and energy which results in confidence and influence. And so nice to see you, Brittany. I remember you, Brittany. Great comment. Okay. So David, let's, so people in the audience, they seem to resonate with Hongik and they see that it's a good vision to benefit all life. So where can we practice Hongik? Is there a specific like training center? Is there like a book I need to read? Is there an online course? Or what do I need to do? But what does it mean? Yeah, to practice this into my life. It sounds great. So as with many things, we can understand it right away and most of us agree with it. You know, yeah, it sounds great. What does it mean to actually expand your consciousness and live more Hongik? I think the key point is, it's about, starts with your mind and body, this idea that Hongik lives inside of us very naturally. It's not some artificial idealistic principle that you need to just think about. So connecting your mind and body, being more present, being able to relax, feel what's inside your body is a good starting point, very important starting point. So meditation, yoga, energy, meditation and breathing, all of these practices can be very helpful. And you got to realize that they're not for the purpose of just being more flexible or even just being more relaxed. They're about connecting to oneness inside, which is where Hongik spirit comes from naturally. I like that, I like that a lot. So where can you do this? Now in some sense, you can do this at home. You can do this anywhere where you can practice with your body. Obviously, BETV, this channel, has a lot of practices for your mind and body, exercise, meditation, movement, all kinds of things. So just by yourself at home, you can turn that on and you can practice along with the video. Yeah, and so going off with what David said is that these practices in brain education, yes on the surface, it may seem like, oh, it's just to make me exercise and become healthy and slim down. Which is good. Yeah, which of course, you need it as the first level of health. But the true reason why we become healthy first is so that my health, my healthiness, my healthness, my state of being healthy, can elevate my consciousness and that I can act from a place that is of Hongik and I can be of service to other people. And very simply, if you want to be healthy, being healthy takes some work. Let's say we're dealing with, we've been working a tough job at a desk and our body posture is not good. In order to change that, you're going to need to work hard for a couple weeks, a couple months to strengthen and fix your body. It's not comfortable to be healthy. It's actually more uncomfortable to do the work to be healthy than it is to take a pill and temporarily feel better without working on it. But if your purpose is, I want to be Hongik, then you can't just take that pill and ignore your symptoms. You have to focus on being healthy and do the work. So it's actually very motivating. And ultimately, when I worked in the body and brain center, now I'm at our headquarters, but in the body and brain center and working with a lot of people, the ones who made the most benefit had a vision of using their body and their life for a bigger purpose, not just to feel a little more comfortable because as I said, working out and improving your body is uncomfortable. So that bigger purpose is what motivated them through the discomfort. So just as you can practice Hongik using the contents on our channel, you can also practice it at any of the entities and places that are affiliated with our brain education practice, which body and brain is another big place and David is in charge of body and brain TV. So that YouTube channel also has a lot of content that to create health in your life, how to practice a Hongik live for creating a bigger change in society, they have those kinds of contents too. So brain education, body and brain, these are two probably the most immediate ways that you can get involved in practice Hongik. That's right. And then you can take those and why not share them with somebody else? Most of the BE practices and body and brain classes are very easy. You don't have to be super fit. You don't have to sweat buckets to get a benefit. You can do some pretty easy. So even people who are dealing with the limitation physically or they don't have a lot of time, you can get a lot of benefit because they focus on your brain as well as moving your body. Right.  Because even if people don't specifically say it out loud, they can feel a change in your energy. They can feel a change in the way because your mindset is different, your consciousness is different, the way that you treat them. I want to kind of mention that point again that you made. It's really important. If you are somebody who wants to help others or influence change in the world, you may have a lot of desperate desire. You want to see this change and you know it's the right change. It can make you very emotional. If you're one of those people who is desperate, I wish, why aren't we doing better? Then I really want to encourage you focus inside. Take some of that energy back and that frustration back and really just let go of frustration about what other people are doing and just connect to yourself. What I found is even a little bit, if you can achieve that, people notice. People start to listen more. They start to work together with you instead of resisting everything that you want. And that can be the difference between mental health and a lot of stress. And it can be the difference between succeeding in your efforts and feeling like nobody's helping. Go inside. And then tell more people about this idea. Because Hongik is something that's very new, especially to Western culture. So maybe even you watching this YouTube live, this is your first time hearing about Hongik. So just as I shared when I just even first heard about it, it gave me a little shift in, oh, wow, I need to take care of myself too because I too am precious and I too am worthy of love. Your speaking about Hongik can inspire that kind of shift, a small but very important shift in mindset and consciousness for the people who are listening to this idea for them to wake up and be like, wow, I am a precious human being that needs care and love too. You got to be willing to... It's interesting because you got to be a dreamer. You have to be idealistic, but at the same time, very realistic. You have to believe that we have this inside because as I said, we're not always connected with that feeling of oneness. Absolutely. Actually, there's a comment from Bill which I think is really interesting. I gave up on pure motivation. Selfish altruism is my thing now. I accept that doing good things inherently benefits me even if that is just feeling good about having done a good thing. It's kind of like fake it until you make it. Consciousness work in a way is sometimes fake it until you make it. It doesn't mean it's fake. It means we start at a certain level and we want to go deeper. We have to believe it and act like it and then we'll feel more of it. Not, oh, I don't feel it yet so I'm not going to do anything yet. It's the opposite way around. I believe it and I take action on it. And then the real energy comes.  How are you like that? Well, I decided and then started to act that way and trust it. And then the energy becomes real. Right. And going off of that and what Bill just commented in the chat is one really big way to practice Hongik is to practice self-care. Self-care. Yeah, practice self-care because Hongik benefits all life. That means the people you love but also yourself. So if you act in a way already that benefits other people in your life, please now shift so that you act in a way that benefits you as well. And a big first step to doing that is to practice self-care. Absolutely. Yeah. Because we already naturally have this mindset and idea that helping others is a good thing. But it takes a slightly different brain cell and a slightly different brain circuit to then think about taking care of yourself to truly taking care of yourself. So what are some ways that people can practice self-care? These days we are so busy. Really? I'm a little busy. I think you're very busy too. Just taking time like sticking to your plan to take 10 minutes for breathing and meditation out of your day or at the end of the day before you go to bed making sure you give yourself 15 minutes to clear and connect before you fall asleep. Just keeping that promise to yourself can be a huge change. And then what you do during that time of course you can do as I said some meditation, some breathing. You can reflect on your day. You can set your vision for tomorrow. Or you can do some stretching to make your body feel better. Those are things that I like to do for self-care. How about you? Yeah, I love taking time in the mornings to come back into myself. Because as you mentioned when we're busy our minds are always outside, outside, outside. So we operate from a place where our mind is not home. And so the way that I practice my self-care and the way that has worked for me is I find some time in my day like you said to come back home to myself. So I do some tapping.  Or just tapping your body like this to bring your mind inside. And that has set the tone of my day way differently than if I just rush up and get up and wash my face and hurry out the door. It really sets the tone for the day to be completely different. That's something that has worked for me. That's really true. If you haven't done that and you think about oh it sounds good but I don't think it'll make that much difference try it. You'd be surprised by how different it feels when you just do that simple thing. That's consciousness different from understanding. A lot of people hear about meditation and body and brain and brain education and they think it sounds good but I've tried things like that before. Don't trust your thinking. Don't trust your memory. Sometimes you had a good experience years ago but then I don't remember what the feeling really was. Try it again. You're going to be surprised at how much it helps. And Maria thank you for your comment. Your comment gave me real joy. So I want to express that to you. Thank you so much. Absolutely. Okay so where can we learn these things David? We talked about some really great gems of wisdom and gems of awakening. So if you feel inspired by watching this what can we practice? You can practice. Well first of all we talked about online with YouTube. You can practice anywhere with BETV and body and brain TV. But practicing in person even if it's difficult sometimes it's worth a 20, 30, 40 minute drive because our brains and our consciousness learn a lot by feeling the energy of other people especially if they are also practicing brain education, mindfulness. We learn a lot more than just through our eyes or more than just reading something or listening to something is by physically being in the same space. So I recommend if you can find a body and brain center near you to go there and meet with one of the instructors because these instructors are people who are practicing regularly every day. And through that their body and their consciousness and energy develop something. It's not about being smarter or better than anybody else it's just about a habit. And a community where we practice these things and ingrain them at an unconscious level in the body. There's something especially if you're sensitive you will learn a lot right away even without words. So if you can check out a body and brain center I highly recommend that. Right and that's where we both started out that's right. Learning about brain education and this philosophy is a body and brain center but if you don't have a body and brain center then brain education TV, body and brain TV is a great resource to start. There's also retreats that you can go on. Right we have retreats at Sedona Mago Center or if you're on the west coast this is in Sedona Arizona. If you're on the east coast we have a east coast retreat center called Honors Haven Retreat. So either one of those could work out well too. Yeah they utilize brain education in their activities and programs as well. Right. So you can find more and more places and organizations and people using brain education around the world but these YouTube channels and body and brain centers and those retreat centers have been doing it for many many years so have a lot of experience and people who are very professional in sharing it. Yeah so can you guys before we close this live today would you share with us what you feel about Hongik? This is your first time hearing about it do you feel inspired to practice Hongik now in your life? If you have some questions that are lingering about Hongik anything that is on your mind about the topic as we wrap up then is there anything else you want to add David before we close? Well while people are typing about their feelings or awakening about Hongik I want to emphasize two things two things the first is keep talking about Hongik keep talking about it and if people come at you and they say oh that's wishy washy idealistic new age blah blah blah what do they call it like kumbaya they're like oh yeah kumbaya whatever it's like okay yeah I understand the cynicism I understand the resentment but where where are we going if not towards more Hongik? What is actually hopeful? Is it just like my country is going to be okay and have more military defense and then anybody who we don't like there's no hope in that yes it's practical but talk about Hongik keep sharing it there's going to be more reality from it and then the other thing that I want to emphasize is don't just talk about it really practice self-care like Linda said with your body take time out get some help meet with other people who practice so talk about the ideal of Hongik and then also practice with your body just do those two things I really like what you said that like really shocked my brain is where are we going if not Hongik? Yeah do we really want to go somewhere else? Right as a society as a world where should we go if not Hongik? What are we fighting for if not Hongik? Are we fighting for going back to the 1890s where every country is just like fighting for itself and taking over other countries does anybody really want that? I don't think so even the people who believe that that's the reality the necessity I don't think they actually want that they just believe that there's nothing else possible when I've talked with them I like that a lot that's going to give me a lot to think about I think that's a perfect way to kind of close with a seed of thought for all of you where are we going if not Hongik? Yeah yeah So let's keep talking about it Yes All right so we're just about to wrap up with this live so thank you all so much for joining and if you haven't done so already please subscribe to our channel Brain Education TV we have more lives like this and more content that talk about the Hongik philosophy brain education how to connect with yourself lots of good good tips exercises and pearls of wisdom that I think are very necessary in our 21st century and then also please check out Body and Brain TV which David is the host of Yeah and then recently those of you here at BETV I think from the toe tapping video you saw a suggestion head over so I saw a lot of you came over and visited Body and Brain TV and saw Courtney Nim's toe tapping video there as well so that's really nice let's keep collaborating yeah so check out make sure you check out like subscribe click the bell for notifications on his channel too because he has really good tips on there as well thank you and let's keep getting together let's keep having this conversation and we love hearing your ideas too yes and then as we mentioned if you can if you live by one please visit a Body and Brain Center because that is the best place to start that's where we started and have grown and and awakened to more of these practices from the Body and Brain Center so if you get a chance if you live by one then I highly highly recommend start there yeah absolutely yeah so thank you all so much thank you Maria Collette, Judy, Yustina Bill, Mukesh Brutney, Semicountry Living oh we got a donation thank you thank you Judy I'm imitating the exercising laughing pair it's kind of it's kind of like an exercise headband right and it's got a little like leaf pony tail so if you want to see us do animated dances then okay maybe it's not an exercising animated pair I don't know thank you so much Judy thank you everybody thank you Linda thank you BTV yeah thank you all for joining today please stay tuned for another live we'll do more lives with David we at least have one a month so please stay tuned for that if Christian's laughing we're glad we can make you laugh and learn at the same time yeah yeah so next time super chat more you're gonna see more yeah huh huh huh unless you don't want to see that yeah unless you send a different emoji then we'll copy whatever emoji that one is okay thank you all right thank you also


Hi everyone, Joe for Jasper's casebreaks.com with our last break of the night. It's going to be 2022-23 impeccable basketball three box picker team number two all-card ship Imagine if all cards in ship and this people be like what what doesn't ship in there? All right, big thanks everybody here for getting in on it We do have another case in the store Jasper's casebreaks.com no filler just yet All right. We got nice Jabari Smith jr. 12 out of 25 For the Rockets that's going to go to brian and houston I hear people pronounce it houston With a u I go with an h How do you enter to win? You can go to jasper's casebreaks.com johnny and buy some spots And then try to win Some cards Hopefully we'll get some nice ones So you can buy spots or teams on jasper's casebreaks.com There's de mar de rosin for the bulls. That's going to go to scott 38 i-75 shading sharp rookie card trailblazers It's going to be for adam kelly And Steph Curry 77 out of 99 What people that's way that What people probably hide people in their classes right? What are you talking about rex? Here's wendelmore jr 22 out of 99 Sounds like you're talking about dastardly deeds Dirty deeds done dirt cheap Nicholas with minnesota And we got jason terry 52 out of 99 Oh people that pronounce houston with a With a u You mean a u right Dallas that's for jt jesson taylor No, no soccer breaks at the moment marco But i'd love to see some someday soon. All right, so we got a couple plate autos here first one is christian brawn Nuggets Nuggets cruise to victory today chris butler with the nuggets to win game one of the NBA finals Then we got a nick jailen brunson 92 out of 99 jersey and autograph That's for new york. That will be for ed p last spot mojo The lat this is a nice one too shading sharp stainless stars That's for adam and the trailblazers No with a u Did i say why maybe i said why But yeah, some people pronounce it houston i for people do it people people in on-air sports media jobs people people not In sports media jobs regular people It must be a regional accent somewhere where some people say houston I say houston hugh houston Well, some people say houston some people say nicola yokich and other people say yokich This one goes to chris and denver The heat are cooked right Nugget denver made it made it look way too easy Heat went on a little bit of a run, but then denver squashed that run pretty quickly I know the feeling That's what happened to my lakers too. Like I felt like oh, there's that 11-0 run here. It comes And then squash It's josh giddy 28 out of 75 okay seat That will be for chris butler We got andrew wiggins 2 out of 49 Wigging out with yuji and the warriors christian wood 42 out of 99 mavs an underrated big man I want to say Well, justin wants to see a one-of-one. Why not? 31 out of 99 bogdan bogdanovich for atlanta that's going to go to dy and the atl Here is ej ladel 45 and a 99 elegance three color patch and autographs a solid penmanship there for the pelz That's going to go to justin for the pelicans And we got marcus smart 43 out of 99 boston's got to retool a little bit I mean that team But jayce they they got a knockout I mean maybe they went sound easy, but you would hope you would think that The expectations are you got to knock out A championship at some point the next What's the window two to three years? There's tyree eason two colored patch and autograph 92 out of 99 for houston bryan with that one and the plate auto is There it is paulo ben caro Rookie on-plate autograph for scott and orlando scott Winning the magic In the team random nine out of 75 Congrats final box good luck. So this is where the case. It should be There we go. That's the uh Silver The big piece of silver there sometimes It could be gold But those are pretty rare. All right 48 out of 49 Valos teammate franz vagner There's your one-of-one chat homegrown one-of-one Chris butler. Okay, see Let's go Chris All aboard the big hit express This might get him in the shop early tomorrow There you go, man You'd be surprised how how well I mean for for some of the top tier rookies like him You'd be surprised how well those do on the secondary market. There's Brandon Ingram for the pelicans for justin We've got walle zerbiak 69 out of 99 t wolves nicolas nice chris and derrick fischer 16 out of 75 Nice one for my lakers eugenie 84 out of 99 immortal ink robert perish Celtics that's going to be for wane and last couple hit or redemption And a paul pierce 15 out of 21 Celtics wane about the Celtics straight up. All right. Good luck everybody We got extravagance autographs j o jordan jordan pool For eugenie and the golden state warriors warriors come out to play All right, and we still got the case hit And the case hit is going to be Oh, one of my favorite lakers pal gasol basketball hall of fame stamp some fine fine silver nine out of 20 lake show Who's got my lakers eugenie has my lakers just retired his number recently Really great speech Really good stuff. There you go ladies and gentlemen. That's the break Let's uh, wow, we still have a wax party invite to give away here as well. So let's gather everybody's names one through 30 new dice new list And name on top gets an invite to the wax party details on jasper's case breaks dot com two and a six After eight name on top after eight one two three four five six seven And eighth and final time after eight A lot of sad faces here, but one really happy face Adam's very close, but it's not quite One really happy face though. A lot of sad faces two through 30. Thank you For giving it a shot plenty of opportunities for we've got plenty of invites left. So keep trying Jasper's case breaks dot com but happy times ultimately for brian Did not call brian's number very often in this break But you got a wax party invite brian. Congrats to you. I'm joe for jasper's case breaks dot com Thanks for watching. Thanks for breaking with us. I'll see you next time for the next one. Bye. Bye


Hello everyone, welcome back for part two of my lecture on integrative modeling of biomolecular complexes, still with a nice background from Pula in Sandinia, and we will start directly after having given you all this introduction about docking in general Haddock and an example of antibody antigen complexes with an example of how you can use data from mass spectrometry experiments to model complexes. And this is the work of Adrien Belkion, a former postdoc in my lab, and Adrien has been working in collaboration with people from the HEC lab in Uthert, Proteomics MS lab in Uthert on the modeling of a bacterial circadian clock machinery. So this circadian machinery of this sign of bacteria is quite intriguing because you just need three proteins, if you over express those three proteins, put them in a test tube, add phosphate, add ATP, the clock starts ticking. Circadian clocks are basically the reason why you will be jet lagged if you were in Sardinia and coming from India or the US for example. So bacteria also have such systems. In this case the clock is regulated at a molecular level by interaction of those three proteins and what's happening is that there is a dephosphorylation process taking place and you can monitor this process by mass spectrometry which allows you really to measure the frequency of the clock. Now at the time when the work was done there was no structure of the full complex known and this is the reason where we try to model the complex and in particular the KB, KC interaction. So what were the data available at the time to model this complex? First of all the stoichiometry of the complex and this was given to us from native mass spectrometry and we knew that KB binds to KC in a 6 to 1 stoichiometry. It means that you need 6 KB to bind to 1 KC. Further there was information about the binding interfaces between those molecules which was coming from HT exchange experiments. I introduced shortly those in my general introduction in the first part of the lecture. So let's have a look at those data. So what you are seeing here is the crystal structure of KB, the smaller of the two components and it's color coded based on the protection factor. So region in blue are protected when the complex is formed from HT exchange and you see two views, rotated views of the same molecule. So this indicates that there is a clear face of KB that seems to be involved in interaction. So that's information which we can give to HADOC. Now the other piece of information that we have next to the HT exchange data are a number of mutations and you see two lysines 74 and 22 and two arginine 74 and 22 and lysine 66 which if mutated abolish the interaction. So this is also information which in principle we can take into account into our modeling. Now this is KC. KC is the largest of the two components. You'll see it's a very large molecule. It has a C6 symmetry. If you look from the top you can clearly see there are six domains. It consists kind of of a double donut. So you see one ring, a second ring. So one ring, the bottom one is called C1. The top one which is called C2. The blue regions are protected from exchange when the complex is formed and you see that we have a problem here because we detect six binding sites at the top, six binding sites at the bottom. So that will indicate 12 to 1 binding possibly but we know it's 6 to 1 and we know experimentally it's possible to distinguish. So we know that the binding should be either at the top or at the bottom but not on both sides at the same time. What you also see is that if you open the complex and this is the interface between the two donuts basically you also see that there is some protection there. So what's happening is that there is some allosteric communication between the top of the molecule and the bottom and this is what is controlling also all this phosphorylation, the phosphorylation process. So we have the information on KB, we have the information on KC so we can define those data as ambiguous interaction restraints in Haddock, do or modeling. We did two rounds of docking, one targeting the top domain C2 and one targeting the bottom domain C1 and what you see here are the top four clusters of those two docking runs ranked and we could not really distinguish if the bottom solutions were better than the the top solutions were better than the bottom solutions. We had a problem here to make a choice which one is the correct solution. So we went to another type of data which is also coming from mass spectrometry and these are collision cross-section data. So just to explain in a nutshell how this works. So the collision cross-section basically is the rotationally average shape adopted by a given molecular ion flowing or flying in a particular gas phase condition. So in the spectrometer, in the MS spectrometers, the molecules are flying through the vacuum against the gas flow of over small molecules and you can think here that the shape of the molecule is going to affect the way and the speed at which this molecule travels through space. So by measuring the time it takes for a given molecule to bridge a given distance in a spectrometer, you can derive information about its shape. It's a long extrapolation because you measure one point which is time and you extrapolate to a 3D shape. But there is information in there and basically you can do these measurements from native molecules with very little samples required. So this is taken so you see these figures are taken from this review Nature Protocol 2008 and if you look at the right graph you see so these are assemblies that consist so these are theoretical assemblies. They consist of the same number of subunits but in different arrangements and you can back calculate what the collision cross section should be from a 3D model and you see that even if the number of subunits is the same in all those the shape gives rise to a different collision cross section. So if we have a 3D model we can back calculate what the collision cross section is and compare that to an experimental measurement and try to use this information in our scoring process. This is what we did for the system so we had collision cross section. You see here the experimental data there's a range indicated by the dashed line so the experimental range is between 133 and 140 square nanometer so it's kind of a surface area that you are measuring experimentally. We back calculated those values from these eight complexes here those eight models with the orange corresponding to the C2 domain the green corresponding to the C1 domain and you see that while the green are all larger than the experimental range and the orange one is fitting at least the top cluster which is a top scoring cluster the best cluster fits nicely in a range. So based on this additional piece of information we selected our top cluster from the C2 domain docking as representing this complex. So this was all mice published in 2014 together with experimental data and then a bit more than two years ago came the cryo-EM structure of the full complex k, a, b and c and basically what the cryo-EM structure shows is that the bottom model C1 this one down there is the correct model. So basically we predicted the wrong model so we screw up somewhere. Well these kind of things happen and in science you're all going to run into things that don't work we're all making mistakes usually you tend to forget about these and don't speak about those anymore but I think it's very instructive and you get educated to show that things can go wrong and also try to understand what goes wrong because in all the experiments that you do and especially when you do simulations you learn most from your mistakes when everything goes right you don't think too much you keep doing the same when you hit the wall that's when you stop and you start thinking what went wrong. Well here what went wrong actually there are several things that went wrong. First of all just let me go back sorry so first of all it's probably not a very good idea to do collision cross-section measurements on non-globular protein if you remember the you see a kc so kc is a protein which has a hole in the middle so it's not globular it's going to fly in a spectrometer in a vacuum and what can happen is that you get a compactation of the system so what you are seeing in the spectrometer will be smaller than what you experimentally have because it's a non-globular protein it means that when you look at the results so this dotted line or dashed line here are an underestimation so if you were to move those up then you will select the other one so that's all the way as a modeler to blame the experiment for our failure but there's another explanation and this is a bit more somewhere worrisome it's nature playing tricks with us the crystal structure that we used at the time for kb was a perfectly fine crystal structure a good resolution at the time when the cryoam structure came a second structure was published for kb and it had a different fold it was exactly the same sequence you see the sequence here and you see the these two crystal structure the the one which is basically called gs is the what they call the inactive tetramerate grand state fold this is the fold that we had at this position at the time and the other one which correspond to the active side active state was more monomeric crystal structure the first part of the structure is the same if you look at the secondary structure the second part is completely different what was alpha becomes beta what was our what was beta becomes alpha so it's a completely different fold so nature is fooling us it's the same sequence which can exist in two different folds so that's a bit worrisome hopefully this is not happening too often otherwise we cannot trust structure biology and and the data in the pdb but from time to time you will have this kind of of weird things happening nature reminding us that we should be humble in our you know in our work basically so now an intermezzo in the meantime we have been improving ad hoc and introducing all kind of new features one of which was to introduce the martini force field into ad hoc to coarse grain molecule the idea of going to coarse draining so martini represents four real atom by one particle or one bead so it's a simplification of the system in terms of number of atoms which allows us to go to larger systems and model larger dimensions they are over additional benefits to do using coarse-graining so if you want to read more I just refer you to this paper this was a work of Jorge in my group a PhD student so basically we go from all atomic model to coarse grain particle we do the docking at the coarse grain level and we transform back the model into a domestic model this is supported now in version 2.4 of ad hoc and also on the web portal so using this approach we went to model the full kb kc complex basically the seven mer complex six molecule of kb one molecule of kc previously we were only docking one kb to kc to one to the domains of kc that were required so now we dock six kb onto kc at the same time using symmetry also as an information because we know that we have this cc symmetry using the same data that we used more than five years ago and targeting basically the same c2 and c1 domains so the top and bottom domain now if you look at the ad hoc score in this case we clearly see that the ad hoc score points to the bottom model as being now the best solution we didn't have this before the docking by doing coarse grain docking we also win in time so we took this model the model is not consistent with the cryo yam basically what you see here is the all model in one color and the cryo yam model in the other one and you see the center of mass of the the 2kb on the cryo yam model we didn't use the cryo yam data to do the modeling but we use the data to validate the model so if we fit our model into the em density and we did that in camera you get a correlation of 0.82 and the model that was directly refined built out of the cryo yam data published in a science paper as a correlation of 0.84 so considering we did not use the cryo yam data into the modeling we get quite a good correlation close to what the cryo yam structure is which is nice so since i was introducing or speaking a little bit about cryo yam now we have a good transition to the next topic in this lecture which is the use of cryo yam data directly to guide the modeling process in ad hoc so cryo yam i think everyone is aware well aware of it but it's really the the new star in structural biology if you go 10 years ago we were speaking of cryo yam as a blobology technique and these days cryo yam is giving us high resolution structure of proteins so what are you doing well it's transmission electron microscopy you vitrify your sample in liquid ethane and then you take images 2d images of your samples and what you see in those images is that you have your proteins that show up slightly the signal to noise of a single image is not fantastic and what you have is images that are of their protein in all kind of orientations so then you have to classify those images depending on your orientation and once you know in which orientation you're looking at your object that you can reconstruct a 3d object which gives you a density so this used to be rather low resolution now it's reaching very high resolution you can also use a different technique which is called tomography where then you are tilting the sample to generate different projections and from the series of tilt you reconstruct a 3d object so again so before 2013 this was a resolution currently we have a domestic resolution it's really impressive and this was made possible by developments both in a software to interpret the data but also in the electronic detection in particular direct electron detectors so if you go into the PDB and the EMDB you will find so this was a distribution of resolution like one year ago and you see now that there is quite a lot of map that are below five angstrom and there are maps that are even below two angstrom these days so it's really impressive it's not like everything is going to be at very high resolution you will even with the current technology you will still have regions in your map which might have low resolution and in the past you have you might have limited resolution maps so resolution is not a constant in cryo electron microscopy that's also different from x-ray crystallography so you have regions that might reach very high resolution but you might have regions when the resolution is still limited not allowing you to build a binissio a model into the data and that's where you go back to the way that the cryo em data were used several years ago was to basically use existing structures of the component of the complex and fitting those structures into the density this was done or this can be done manually using tools like a usf camera there are also software that can do an automatic fitting into the map a different version of it power fit being our own when you are doing this fitting typically what was happening is that you fit one component at a time into the map but you don't consider the interaction the energetics of the interactions between those fitted model meaning that interface are never really optimized the model looks like nice when you represent them but you should not turn on the sidechain otherwise you're going to realize that there are a lot of clashes experimentalist might tell you that anyway you should not look at the sidechain if the resolution is too low so that's that's a debate I think if the model is to be useful it should also make sense in terms of quality and stereochemical quality so this is something we wanted to look at to try to use this this map information cryo em density as a restraints into haddock and this is the work of a hido former phd student in a group we implemented basically em restraints into a doc to guide the docking process so haddock is using cns as computational engine crystallographic nmr system it stands for and cns was originally designed to do crystallographic refinement and it has a lot of tools for nmr as well it's scriptable and all of haddock is written at the script level in cns so cns has from the start all the energy functions to handle x-ray density maps so what we can do is transform or cryo electron density into a format that cns uses understands and then use the restraints in cns now if you try to turn the em restraints directly from the beginning of your docking and try to dock your molecule into the back what we quickly realize that the solutions are not converging so instead of using the map from the beginning to to drive the docking what we are doing is to identify centroids so the most likely location of the molecule into the map so we define those centroids and then we use distance restraints to guide the molecules toward those centroids now if you don't know where the molecule are going you can define those restraints as ambiguous which is very natural to what haddock is doing you do your energy minimization using the distance restraints and once the molecule are then into the map you turn on the density and optimize against the density and once this density is there we can take this through our refinement flexible refinement stage and also it's part of the scoring function so first centroids to pull the molecule into the density once they are in the density we turn on the density and refine against it now how do we get those centroids position you could get that from usf camera but because we needed them we also started looking into our own fitting software to to systematically fit molecule for all possible translation rotation into the map and this is what gave rise to power fit and power fit has been adapted so that it gives us the position of those centroids which you can give to haddock now here let's look at at the real case so this is the integrative modeling of the 60 and s ribosome and ksga a protein that binds to the 16x ribosome so what were the data available at the time so there was a 13 angstrom resolution cryoem resolution map available a crystal structure of the 16s ribosome was known there was a crystal structure of the protein the binding site on the ribosome was mapped mapped by hydroxy radical footprinting so we we know from this information where the protein is binding on the ribosome and there was also some mutagenesis data on the protein side like those free arginine that you see here that if mutated basically prevent the binding so this is all information that we cannot use in haddock to to look to refine this complex now there's of course a model of the complex that was deposited at the time it's entry for adv it looks very nice as long as you sure need a backbone but as soon as you turn on sidechain you see what's happening here the interface between the protein and RNA is full of clashes and if you concentrate on what are those three critical arginine or residue doing one lies into arginine this one is not doing anything it's not contacting the RNA this one is clashing and there's only this lies in that kind of makes sense so the model which is there is not explaining basically the data that we have so the question is can we do better so we're going to use haddock to try to debump this model and improve the quality of the model and this is the result of the protocol that I just showed you so we put all the data together we do our energy immunization with centroids we turn on the density we refine the complex and you see here this is rmsd from the the cryo-m model versus core we get one set of solution and then you can zoom in again at the interface and now you see that all those amino acids which were known for mutation to be critical they make interaction that makes sense it doesn't mean per se that they are correct but at least it makes sense and what is also interesting is that the model reveals additional residue that could possibly be critical for the interactions for example 147 and 248 are interesting arginine they have not been mutated but this will be a validation so we should mutate those test the binding and see if the model is indeed predictive or not if we look also at the sequence conservation we see that all those arginine that we find as being important for for the interactions are indeed also highly conserved so there is some backup from evolution there that indeed there is something interesting happening about those residue so in conclusion for this the use of cryo electron microscopy data into haddock so they are now fully supported it's part of haddock version 2.4 it's supported also in the web portal the new web portal 2.4 of haddock so we use distance restraint concept to drive the docking and initial and the first stage and then we turn on the density to refine and optimize the model it's map size independence you don't need to use all the density you can basically take a take a subset of the density map where you are interested in and it's fully compatible with all the other type of restraints that are supported by haddock so it it can be used in an integrative way to do integrative modeling so this work is also published in structure so you can look up the paper if you want to know all the details now the next topic so we spoke we've been speaking about very large complexes 16 ribosome and these bacterial circadian clock machinery so now we're going to zoom in into more smaller and molecules and we're going to look into drug like molecule so haddock has been pretty much from the early days supporting small molecules we first implemented support for cofactors because there are many proteins that have cofactors and those might be important for the interaction as well so the haddock server which exists since 2008 has from the start supported small molecule and we have been doing some work in the past also in you in the docking small molecule but we have no ambition to compete with all the very nice small molecule docking software out there like no glide autodog vina and this kind of molecule but one feature of haddock which makes it interesting is that we can define specific distance restraints between two points between two atoms and a lot of the small molecule docking software could not do that until quite recently and this is the reason why for example companies like zo bio which you see in this publication I've been using haddock since quite some time to drive the modelling of protein ligand complexes based on nmr data that they could measure so they have a technology when they can measure a few distances between the ligand and the protein and they use that for for basically guiding the docking at the time the small molecule docking software could not do that they will only be able to filter another example more recent which is coming from Novartis jack's paper 2017 for the same reason using the classical small molecule docking software they were not getting complexes that make sense of their data but using haddock they could define specific distance restraints generate models for system which would not crystallize based on the models do structure based drug design improve the models improve their design and then those started crystallizing and then when they compare the the crystal structure with the prediction for haddock they were coming nicely together that's that's a nice story we are not involved in this work but it's a nice publicity for haddock and small molecule now a few years back we discovered this drug design grand challenge drug design data resources and so d3r and d3r has been running the blind experiments that test the performance of small molecule docking software and push the boundaries of what is doable basically in drug development so we participated to three such grand challenges two three and four those are organized in two stages in the first stage you have to predict the poses predict the structure of the complexes and rank them and in the second stage crystal structure are released but then you have to predict the affinity of those of the of the molecule one good thing here is that's all the for each challenge basically for one set of complexes they are all coming from one specific company and it changed which company is providing the data but they have all been measured in a consistent way so they might have ic50s or binding affinity data which have been measured by exactly the same settings which is very important so this is this work our participation d3r has been mainly done by panos former phc students and then a former postdoc in my group so the first target that we encountered if we are was the phanezoid x receptor fxr also known as nuclear bale acid receptor so it's a pharma target involved in the diabetes among other so the challenges in this system are they are twofold first of all the binding site is quite buried so it's a hydrophobic pockets the ligands that are existing that are known are branch ligands so you have a buried binding site so for us for haddock this is going to pose problems because when you do small molecule docking for example of autodoc you build a box around your binding site and this is the only region where you search now in haddock we start with the molecules separated in space and they will need the small ligand will have to penetrate through the protein to reach the binding site the other challenge is that there are quite some conformational changes in the protein depending on the ligand to which it's binding and you see here this is a post d3r analysis of the different conformation of a receptor bonds to the different ligands that have been crystallized and you see that indeed these helices that are on top of the binding site do show quite some conformational changes depending on the ligand bound to it so you need to to be able to to describe these kind of changes to some extent to get reasonable predictions so what did we do you are given smile strings so this is a text string that describes your ligands so first we generated a 3d conformation of the ligands using the open eye omega toolkit which is freely available for non-profit we clustered those solutions and selected a representative from the different cluster and then we're going to give an ensemble of conformation of the ligand to haddock so this is this implicit flexibility treatment i've been speaking about in my introduction so this was a ligand site for the protein site we searched the pdb and we found quite a number of structure that had been crystallized already with ligands we cluster all those structure based on the binding the similarity of the binding pockets and then we selected cluster representative so this gives us also an ensemble of structure for the protein so we're an ensemble of structure for the ligand we have an ensemble structure for the protein we defined the binding pocket as all the active residues that are within five angstrom of all the ligands that were found in the that were available in a pdb and we did our docking and when the results became available this was the first two ligands i started looking at and if you look at the the left side so they say well it looks like the prediction was not so bad we were 1.2 angstrom away from the crystal structure i say oh that's quite nice so properly protocol is not doing so bad here you see that they are a bit more so this is two angstrom the limit in small molecule docking for acceptable solution is around two angstrom so you see here we have flipped a group here so that's why the harm is this is much higher so we thought okay probably we didn't do that bad in this prediction when the results came out the green bar is where we are standing so you see that the average actually of the best prediction so we can submit five predictions per ligand so the average of the best prediction in the top five for us was above five angstrom so we did the protocol completely blindly we didn't even even look at what we submitted in most cases so some ligand probably even didn't penetrate properly the the binding side so this is one of the limitation okay so that's another example where basically you cannot hitting the wall so yes we didn't do that well so what went wrong so this is when you start learning your lessons so first lesson but also by participating to the d3r meeting and talking to people is we have to do a smarter choice of the receptor conformation okay second lesson we need to select also the ligand conformation much better for the docking process and not use of this large ensemble that we did before and the third lesson was that the starting point which is randomly separated molecule in space is not ideal for small molecule because we have this problem of reaching the binding pocket well this is all they are all good good reason why these are our main limitations and you can read all the details in that paper then came basically grand challenge three which is not katepsin against 141 small molecule for 24 of those we had they were crystal structure so we had to predict those 24 structure so to select the receptor conformation now we compare the ligand that we have to dock with the ligand that are present in the crystal structure available in a PDB okay so we need a template in a PDB in order to be able to do this and you see here so we're going to select the receptor which has the ligand the most similar to the ligand that we have to dock okay so and we are for calculating the similarity of of the ligands we use the tanimoto coefficient which basically compare the similarity of substructure in the ligand so we have a smart choice of the receptor now for the ligand we still generate a lot of conformation up to 500 using open eye omega software but now we're not going to do clustering and select all those conformation but we are selecting conformations that have similar shape and also kind of chemical properties as the one found in the receptor that we have selected in a previous step so we can do like shape comparison of the ligand this is implemented in a rock software in open eye and you see here so these are different ligands but you see that they have rather similar shape okay so by doing this approach we're going to select the tan ligand conformation that are the most similar in this comparison to the template ligands which we have found from the from the receptor we selected and this is what we now going to give to haddock the third lesson in gcr2 was that our starting point was not ideal so instead of doing the full docking process in haddock we're going to superimpose the selected conformation of the ligands that we have to dock onto the crystallographic ligand in the template that we selected and we use for this again open eye software called shape tk so basically now we're only doing the refinement the final refinement stage of haddock in final solar okay now this is the the set of solutions so you see here the 24 ligands this is the rmsd from the crystal structure so these are already the results so you have 2.5 angstrom here and you see that in most cases we have predictions that are below 25 angstrom so these are some of those so this is below one angstrom very nice prediction here is another one 1.75 angstrom these are quite large ligands you also see these are the top five poses you also see that the scoring function is doing quite well so you see so this is one cluster of solutions this is another cluster so we still have this cluster base approach and you see that the top cluster is actually the correct one same story here same story here so in most cases you see here top cluster correct solution so the only few cases where actually the order is reverted so scoring function is doing quite okay so where does that put us in uh overall performance then you see now that we are done here at the top of of the predictors the top is quite flat actually so this is position six and we have about two angstrom median over all poses so that's a very nice result so we have learned all lessons from grand challenge two applied those in grand challenge three still without knowing what they will but the outcome will be but it worked clearly nicely uh grand challenge four came in 2018 and yet another protein beta secretase 20 small molecule for which they were crystal structure we follow exactly the same template based strategy as in gc3 these are some of these are our predictions superimposed onto the crystal structure one of the challenge here was that the ligand was cyclic and you see again that we are doing a very good job at predicting the right conformation the median or best model at 1.2 the top one model 1.5 and the top five 1.7 uh using exactly the same template based approach as in gc3 and this is all described in this paper published in 2019 so where does that put us against the entire list of participants and in this list these are not different participants they are also the same participants trying different approaches and you see that we have done three submission this was a template based approach and here we have been trying some other things but again you see that we are doing very well around one and there are groups that are going as low as 0.5 or more so i think so so this is this template based approach clearly uh makes sense so in conclusion for this d3r uh challenge uh this kind of challenges but also capri for protein protein complexes are very much catalyzer in terms of software development and methodology development because you learn from your failures you don't learn from your successes usually you learn from your failures the success factor for small molecule docking for us was really to to follow a template based approach small molecule docking which consists of a smart selection of the receptor conformation and the ligand conformation for the docking and also a smart positioning of the ligand in a binding pocket and then just refining the the pauses now this brings me to uh to one of the last topic but uh looking at the time now for for this uh for this lecture i think i will skip uh this topic i will record it as a bonus session which you can look at at the later stage if you want so i'm going to go directly to the conclusion and perspective part so in conclusion i hope that i've given you an overview of integrative modeling what it means and in particular what how ad hoc works under the hood what it can do i gave you several examples of using different type of data to guide the modeling process what we always have to realize that what we are doing when we are doing modeling is generating models they might not represent the truth they might not be perfect but they provide working hypothesis and in that way they are still useful so if you take the model and then generate hypothesis go back to the lab or talk to your collaborators and if you can generate do experiments metagenesis some of our experiments to validate the model then they are very useful because they are driving the experimental work so modeling is not the end of the road modeling is just the start from your experiment so you have really to see modeling and the experimental work as intertwined and you go in circle and using data when you cannot solve the structure in a classical way is really a very complementary to classical structural methods and you will see more and more of integrative modeling coming out this year the coming years there is even no database which is accepting integrative model called pdb dev now do you want to learn more there will be a practical tutorial in this online course but we have also many more tutorials that are online so if you visit bonvanlab.org slash education you will find tutorials for version two two which is the old version of haddock but the two four version some of them some of those are quite complex tutorials the tutorial that we're going to run in this thermal school is not yet in this list so you will have a primer in terms of tutorials now to finish i want to of course thank the people who are doing all the work i have the pleasure to give these lectures and go to nice places like pula virtually this year so all my group over the years so this is a picture taken about a year ago not everyone currently in a group in the picture but it's a good representation of the current group members which are also all listed on the bonvanlab website and we could not do all this work if it was for the funding of several national and european project with bio excel being very important to catalyze and support all the software developments around haddock with this i'm finished i want to thank you for taking the time to listen to me online you can find much more information about what we are doing our research and our software at the bonvanlab.org site if you have any questions about ad hoc we have this ask bar excel forum and with that i thank you again for your attention bye everyone and hopefully we'll meet sometime some place in real life