Hello everyone and welcome to our webinar. I'm Rostin Apostolof and I will be today's host. This is the fourth webinar from the BioXcel Educational series and today we will be having a look at some automation methods for free energy calculations using a tool called BMX. And it will be my great pleasure to have today Berthegroth and Vas Jager to present this material. Before we start I have to make a few announcements. First you should know that this webinar is being recorded. We will put the recording on the BioXcel YouTube channel and also on the website so that you can watch it later. At the end of the webinar we will have a questions and answers session where you can ask questions to Berthegroth and Vas. And you can ask your questions using the goto webinar control panel. There is a questions tab there and I will give you the microphone to talk directly. If you can't, if we have problems we call you, I will read the question on your behalf. Before we start I would like to give a very short overview of BioXcel which is the organization that is organizing this webinar series. BioXcel is a center of excellence for computational biomolecular research. It is established in funding from the European Commission and the center works in three main directions. One is towards improvement of performance efficiency and scalability of three key software packages that are widely used by the computational biomolecular research community. One of them is Gronk probably many of you are familiar with it. It's also the backend behind PMX that we presented today. The other application is Hadoq for integrative modeling of protein complexes and also small ligands and we also work with CPMD specifically on QMM approaches to modeling enzyme reactions. BioXcel is also working on devising efficient workflows including data integration. We work with several popular platforms and workflows such as Galaxy, Ignite, OpenFacts, Pashita, Verna, Comps you might be familiar with some of them and the center is developing an extensive training program. It's providing consultancy services to promote the best practices and make all users of those applications do their work better, faster and more efficiently. What might be of interest to you is that we are launching several interest groups on certain topics related to computational biology and some of these interest groups might be interesting for you to join. We are just starting with them that you can visit our website bioxcel.eu slash interest dash groups and you can subscribe to those mailing lists. BioXcel provides various forms of support. We have forums on ask.bioxcel.eu, we have several other ways of contacting us. The chat channel is also open and we have a video channel where we have copies of the webinars and we will be putting additional material in future as well. Now without waiting I would like to introduce today's presenters. We have Professor Bern de Groot from Max Planck Institute who's been working for many years in the field of computational well molecular dynamics simulations. He's working extensively on chromax and free energy calculations. We have also today Vance Jager who is a postdoc in Bern de Groot's group and they will tell us more about how we can automate the calculation of free energy and we're looking forward to their presentation. So welcome, Bert and Vance. I would ask you to change all the presenters to them. All right. Thank you very much, Rosen. My name is Bert Groot and together with Vance, as Rosen already said, we're going to spend the next 14 minutes or so talking about how one can do free energy calculations in Gromax utilizing the BMX framework and I should really say that this is to work primarily of phytotoscapsis who together with Vance works on the development of BMX and this all started with the work of Daniel Seliker. All right, so before we jump right into it, maybe a few words on why we would be interested in free energy calculations and particularly also of free energy calculations on protein mutations. Why proteins? Well, proteins are nature's nanomachines and they carry out most of the functions in our human bodies and also throughout nature and as such it's not surprising that actually many diseases are linked to mutations that occur in proteins so that's where a major interest into protein mutations comes from and the other key motivation to be interested in mutations is also because they are really at the heart of protein engineering and design so to be able to use proteins for for example industrial applications and so why would we want to do free energy calculations? Well, as you probably know, affinities are free energies, free energies of binding and that means that the affinity for a ligand binding to a protein or a drug molecule binding to a protein the affinity is given by the free energy of binding and the same holds for two proteins binding to each other so in the field of protein protein docking also the binding free energy determines their affinity. Another form of free energy is stability so in that case we talk about the free energy of folding and their folding free energy gives the stability of a protein for example against a denaturation or a thermal unfolding so again there also if we're interested in how a mutation affects the stability of a protein we might be interested in computing free energies and if we want to do that on a on a large scale then of course it would help very much to have an automated and user-friendly framework to carry out those calculations accurately and this is exactly where PMX comes in. Now a little bit to give you a sort of a background where where PMX fits in of course there are many different methods to calculate free energies ranging here on the right hand side from fully statistical approaches like docking for example to more hybrid approaches like PBSA calculations or also Rosetta falls into that category and then on the left hand side we have methods that are based on the first principles of statistical mechanics and molecular dynamic simulations would be among those methods and and among those we can again distinguish between two different flavors one would be free something so if we are lucky enough that the process of interest takes place on a time scale that we can easily simulate say for example we have the reversible folding of a protein or we have the reversible binding of a ligand then we can just count how frequently are we in a folded or in a bound state and compute the free energies from that but in most cases we're not that lucky and we need some sort of biased sampling and then we can go to enhanced sampling techniques like umbrella sampling that is based on an equilibrium approach or we can also use non-equilibrium methods like Kersinsky and the other branch of bias sampling is comes in by a so-called alchemical methods and that's exactly the topic for today now why would we want to work with an alchemical method but the example here shows how that can be useful if we are interested in the binding say of a ligand to a protein here on the top and experimentally we would determine the affinity by just computing the delta g of binding of this ligand to the protein now in a simulation this might work but this might also be problematic yeah if the affinity for example is high then it might bind but not spontaneously unbind or vice versa so we might have the difficulty sampling the pathway in a way that we can extract the free energy in an accurate way but if we are interested primarily not just in the affinity of this ligand but rather in the question well what if we change that ligand how would that change the affinity then we can apply a very neat trick and that comes from this thermodynamic cycle and so there the the question is about the difference in affinity between these two ligands on the left and instead of then binding to the protein in the simulation and computing the free energy change what we instead do is we change the ligand from its initial state to some modified state here we add a methyl group to the aromatic ring and we can compute free energy for such a process and we call that an alchemical transformation because really what we're doing here is we are generating atoms out of nothing and we can of course do that in the unbound state here on the left and we can do that also in the bound state on the right and we can compute the free energy for that and now the nice thing of thermodynamics is that it's the free energy is a state variable and that means it doesn't matter how we go from say the upper left to the bottom right if we go via here or via here and we always get the same free energy value independent of the path it also means that the difference between the two black delta g's and so the two horizontal transformations must be exactly the same as the difference between the two green transformations and that's exactly the trick that we apply so what experimentally one would obtain as the difference in affinity from the delta g3 and delta g4 is what we computationally get as a difference in free energy in morphing in the unbound state versus morphing in the bound state um as oops a similar thermodynamic cycle is what we can apply if we look at protein folding so again the folding process by itself now as a as a vertical arrow might be problematic because it's um it might take milliseconds or longer for a protein to fold so we might not be able to access the um the folding or unfolding pathway in any reasonable way and compute in associated free energy but again what we can do is if we are interested in for example how a mutant effects the folding free energy or instability we can make an alchemical transformation of one amino acid into another here from bloom to red and we can do that once in a model of the unfolded state and once in a model of the folded state and again the difference in the alchemical free energies is the same um by definition as a difference between the folding free energies of the native protein versus the um mutated protein now how how do we exactly compute the free energy across such an alchemical pathway we do that by introducing a parameter usually called lambda that we um that is a coordinate that we switch from zero to one where um it is zero in the starting state and it is one in the target state usually these two states are referred to as A and B and um we then get access to the free energy of this process of switching from A to B by integrating um lambda from zero to one over the um a force that is acting on lambda or the derivative of our um potential energy function with respect to lambda and we can do this either at intermediate steps or lambda that we each um simulate in a discrete manner that's then called discrete thermodynamic integration we can also let it grow um successively that is then called slow growth thermodynamic integration um the emphasis here is on slow because the assumption underlying all this is that during this whole transition process the system must be in equilibrium so that means we should be switching slow enough that the system is is always in in equilibrium um this gives us access to free energies of these alchemical changes and um maybe some of you are wondering well how can it be that this is now a free energy where we just said that we are just integrating um across the gradient of the potential energy function um and nevertheless I'm claiming that here we have also the anthropic contribution to the free energy I think this is a nice piece of homework and um I think many of you should be able to figure that out if not please feel free to contact us at any time okay I was um emphasizing this requirement of being in equilibrium actually um there are workarounds if we um do this switching between a and b so fast that we cannot assume to be in equilibrium anymore and this is based or one way of doing that is based on the crooks fluctuation theorem so now if we have a if we are switching between states a and b both of which for which we have an equilibrium ensemble with thermodynamic integration we would need to do slow transitions between the two and get access to our um free energy if however we switch fast then we get um the free energy difference plus an additional contribution due to dissipated heat so we get something different from the free energy and um in fact if we do a um a number of these fast transitions we get the distribution of such work values non-equilibrium work values and the same holds for the um backwards transitions and so we get the distribution of forward and backward um work values and the crooks fluctuation theorem actually tells us that where the two um are identical so where the two probabilities are the same this is where the work values give us the um the free energy value so that's exactly at the intersection point of the two distributions um so applied back to our thermodynamic cycle of such um alchemical transformation this means um that we um um do that more thing once for example here in the non-bound state and once in the bound state and the difference um gives us the delta G that we're interested in um now um that was a bit of a yeah free energy calculations alchemical free energy calculations in a nutshell how does PMX help us with that um well um what you may have noticed is that um if you are familiar with MD simulations if we talk about the state A and the state B between which we um want to make a transition of course both will have a different topology so a different um molecular descriptor with different bonds and angles and atom types and what have you and this is something that um molecular dynamics engine has to know about of course if we want to make that transition um so in the gromax topology for example you can define an A state and the B state and this is actually um quite tedious to do if you want to do for example a um amino acid mutation yeah here we have um the generation of an aromatic ring that includes quite a few additional um atoms and and interaction parameters and now what PMX provides as a functionality is that for any mutation you might be interested in it gives you the framework to generate um such um parameters automatically so we have a mapping of each amino acid into each other amino acid except for proteins um and so we don't have to um generate these these mutation topologies by hand anymore um so within back of our thermodynamic cycle did now this now allows us to do a whole set of um mutation free energy calculations well as as one of the first sanity checks to see if the software works as it should um we did the number of um um at trivial cycles where we mutated um evading for example here to a phenylalanine and back in the same box as a two peptides one that was um simulated in one direction one into the other and um if we then um um make the calculation of course um a zero should be the results yeah because the beginning and end states are the same and um and and and quite a few errors in in creating the topologies and so um would occur in these free energy values to dba from zero so this is the first um check to see if things work as they should and we see here actually for quite a few mutations um even the changes where the charge is not conserved in quite a few different force fields we all get the scatter around zero so this is one sanity check um that appears to be fulfilled now of course the real um more interesting check is to compare against experiments and one thing that we were interested in is to see how well would modern day um molecular dynamics force fields um be able to predict changes in um thermosability of proteins uh due to mutations and for that we looked at um primarily at the protein barnace for which there is extensive um data in the literature and we looked at in total 119 mutations at 55 different positions and um we have data from calorimetry available so this allows us for a direct um assessment of the accuracy that we can reach with these kind of calculations um we um chose five different force fields that are popular so two amber flavors two charm flavors and opls and um we used the crooks set up that I introduced a second ago with 20 nanoseconds of equilibrium for each mutation and then 100 fast transitions in both directions which were really fast only 50 picoseconds each and we used PMX for all the topology generation as an overall accuracy um we get quite a reasonable correlation with experiments so here we have the experimental delta delta g for um these mutations versus the ones we calculated using the charm 36 force fields so we get an overall reasonable correlation with an um a mean unsigned error of about um slightly less than one k cal per mole and actually we find this for most of the force fields that we looked at so this charm varying does a little bit better than all of the others opioles seems to be um sort of on the maybe slightly uh doing slightly worse but overall one can say that on average we're sort of in the bulk part of an accuracy of about one k cal per mole um of course with that statistics we can also see where this error that we have where it comes from and I don't have time to go into that in detail now but we notice actually that we have a similar error in the calculations um one is a systematic error that comes from the force field one is a statistical error that we have from the something with the settings that we chose and the third source of error is experimental error and that's actually something you can see in this scatter plot over here this is experiment versus experiment and we also there see um for the same mutation in the same protein quite a good correlation but also quite some scatter so this also um leads to an uncertainty if we compare simulations to experiment what is also means means since we have a systematic force field error um we thought well maybe it helps then if we combine the results from multiple force fields and that's what we see over here so this is the individual force field results that we had before if we now take um a consensus approach taking results from multiple force fields we see that indeed we can improve quite a bit on the results of each individual force fields depends a little bit what consensus approach you take but even just um averaging two individual force fields already help so if you have the possibility it's always good to to compute things in multiple force fields and then take a consensus approach um of course we were interested to see if this not only works for barnage but also for other proteins so here we have an example on the Staphylococcal nuptias and a GPCR so membrane a receptor and we see that in both cases so for the nuptias we get a mean error again that captures around one kcal per mole for these different force fields which is similar to what we found for barnage and for the neurotensin receptor we don't have experimental free energy values but we have thermostabilites in terms of melting temperatures and also there we get a positive correlation between our predicted free energy changes due to mutation and the experimentally observed melting temperatures. This is where I would like to switch to Vance Jäger who's a postdoc in the group and he does the PMX calculations on a regular basis so he's in a much better position to talk you through how PMX actually works in practice. So we'd like to present to you a practical walkthrough of how one might conduct these simulations. I'm going to show you the four major steps of setting up and running an alchemical mutation and how to analyze the results using scripts available within the PMX package.  First let me describe the protein that we're going to be mutating today and it's this tripe cage protein. Many of you are probably familiar with it. It's 20 amino acids long and it's the smallest polycoptide form a stable folded structure so for that reason computational studies often use it. What we're going to test today is whether or not we can substitute this central tryptophan residue for a phenylalanine residue and see if we can predict whether or not it will still form a stable fold. So the first step as Bear was mentioning of a now chemical mutation simulation is to generate this hybrid structure in topology and PMX is perfectly suited for this task. On the left you see the atomar structure that you can get from the protein data bank and on the right is this hybrid structure that we would like to construct and the hybrid structure is going to contain the side chain of both a tryptophan and a phenylalanine so if we set lambda equals to zero we have only a tryptophan we set lambda equals to one we have only the phenylalanine and then values in between are some portion of each. So I'm going to first show you how to generate these hybrid structures in topology using an automated server that we've recently set up in our group and then I'll later move on to show you a manual method if you want to script out some of these simulations for a wider range of mutations. So when you first arrive at this PMX web server that we've created you'll be greeted with this page and within a few simple steps you can transform your protein. So first what we do is we upload a PDB this PDB here is the the first model structure of the of the NMR structure for the strip cage protein we select the amber 99 SB force field we can use we can mutate the protein multiple times at once but in this case we're only going to mutate it once and we're going to change amino acid number six that central tryptophan to a phenylalanine and we suggest usually we use this PDB to GMX to assign hydrogens because if there are mismatches between your PDB structure and a naming convention within these force fields you will get some errors so we tend to use PDB to GMX to assign those hydrogens. So from that we can submit our submit our job and the next the next next screen you'll see is a confirmation that your job has been submitted to the server and it should only take about 30 seconds or so to complete that depends on the size of your system and how busy the server is and after that you can click on on the link on the link given here. So here is where we can download our results and the files these files the server will be cleaned occasionally so we suggest you download the files here on the machine and not rely on the link. So the two files that you're going to get out are a hybrid topology and a hybrid structure and what you'll notice here is at the bottom of the warning that says do not forget to set the GMX load variable to the mutation force field 45 directory and that'll be something something important later on otherwise Gromax Gromax will get confused if it doesn't have this GMX load variable set to this particular force field. So here are the two files that you get out from the web server they're on the left you see your hybrid structure on the right you see your hybrid topology and we know that the mutation is taking place and that it's correct because we have a column here that has a W2F and that means that tryptophan has been mutated to phenylalanine but if you are familiar with yeah so that those are the two files that you will get out of the automated server and now that I've sort of shown you how to automatically do this the easy way to do it I will show you a little bit more difficult way to do it the manual way to do it and this can be useful for people who want to run a large campaign of simulations for many different simulations for many different mutations so here's the manual or sort of method first what we'll do is pre-process the structure so we start with this one one l2 y pdb and we run it through pdb to gmx in this case we're still using an amber 99 sp force field and tip 3d water and this should be no problem for anybody who's familiar with Gromax and when you download pmx you'll find a folder of scripts that allow you to manipulate the topology and the structure files and eventually I'll show you later on how to analyze the results you get from the simulations so in this step we're going to use one of those one of those scripts called mutate.py to change our pre-process structure into a hybrid structure upon executing the python command shown here you'll be given the option to select your mutations here we select residue number six obviously and retain it to funnel islanding and we would not like to apply another mutation even though you know if you would like to you can apply multiple mutations you can also use this flag minus script if you would like to script out multiple mutations and you can you can look in the help menu of mutate.py if you if you'd like to do that there's more information in there so here is our resulting hybrid structure that we generated manually it's the same structure as we got out from the from the web server as you can see the w2f residue name here so what we'd like to do next is is create a topology file and this yeah so we're going to use the hybrid structure with p2p to gmx to create a topology file that contains the hybrid residue and let me remind you once again about this gmx lib variable if it's not set correctly you will get problems so after this if you're familiar with pb to gmx you will get a topology file out but that topology file that you get out will need to be edited so if you're familiar with alchemical mutations and you're familiar with chromax files this topology file is missing the the b column so it does not have the b state that we need and in order to create that b state we're going to use a package called generate or a script called generate hybrid topology and with the script we input that topology file and we we will receive back topology that includes that b state so i will show that here so here we highlight new topology file and you'll notice in and read the additional information added this b state so these steps that i presented here in the last few minutes this is how you manually set up the structure and topology and if you're not interested in doing many multiple different mutations likely the web server is enough for you to do what you want so after we have this topology and uh after we have this topology and structure we're going to minimize the pull rate and mutate the the uh the structure so for those of you that are familiar with bromax next few slides should be easy to follow we have simple mdp files available linked at our web server page uh so to go to go over the setup and minimization quickly first we added the box size and shape to make room for the solvent this is going to be a cubic box with 1.2 nanometers of buffer between the protein in the edge of the box and next we use gen box to fill the box with water we generate a tpr file with gromp that we then neutralize and ionize and then we can run our minimization and that minimization should be very fast for a small protein like this it should shouldn't take much time at all the next thing that we need to do is equilibrate the box in order to generate an ensemble of structures from which we'll we'll do our alchemical mutations where we drive lambda from zero and one and also drive lambda from one to zero to achieve the mutations so the important portions of the mdp file a few of them are labeled here in red you can set your initial lambda to either zero or one depending on whether you want to equilibrate for your a state or your b state and then you can set your delta lambda here that's how fast lambda changes to zero such that it stays in state zero or state one for a protein of this size we um yeah we're going to run the equilibration two separate folders and try to get about 10 nanoseconds of equilibration and we're going to use frames from the last athlete trajectory pull them out using trash can bear and once we have those frames and we're sure that they're that they're close to some equilibrium we're ready to mutate here are some of the important parameters that we might change for our simulation we need to calculate the energy every step that's dh or d lambda as dh d lambda every step as well as calculating the energy every step um and then we can also change how quickly we we mutate between state zero and state one so that's the number of steps in our simulation we can change the initial lambda either from either zero or one whether we're starting from the a state or the b state and the delta lambda is how much lambda changes with every time step so the more if you multiply delta lambda times and steps that should equal one or negative one some of the parameters that you might change within your simulation campaign or the number of transitions that you have and even the analysis methods at the end and and i'm going to show you in some future slides how changing those parameters might change the results that you get out so here's what you will get out after you do an alchemical mutation simulation this one we ran for 50 picoseconds and and what you'll get is this extra file called dhdl.xvg and as uh as barricade shown before we can get a work a work value out from integrating this integrating this dh or d lambda uh over the whole range of lambda and from that we can get a work distribution and we can begin to analyze we can begin to analyze what the delta g is for this mutation so here is here is another script that we have within the pmx package called analyze crook stop line and this calculates the delta g of the mutation using several methods and here's the output file it's called cgi.png this is one of the output files from this analyze crook stop line we can see the work distributions for the forward and backward transitions in red and blue and the intersection of the two Gaussian curves fits those distributions gives us an estimate for delta g using a method called the crooks Gaussian intersection method some other files that the script will put out are this results dot that so this gives you information about uh different statistics about how well the how well the fits the fits are as well as it analyzes using two other methods which I didn't actually print out the results the red dots there there there will be results in yours using the benign acceptance ratio and the jarzinski estimator so now that I've shown you these files I'm going to show you just one sort of check as to how you can determine whether or not your simulations are well converged this is one more file that comes out called w over t dot png and what you can see on the left is the work value the left part of this graph is the work value over time and if you see that the work value fluctuates around some central value rather than uh rather than than skewing one way or the other um or or having a large jump during your simulation you can assume that at this state um yeah you can you can assume that the the state that you're simulating here is actually decently well cooperated um so here what I'm showing you is are some of the differences uh what happens when you use different transition times during your simulation so this these are these are the same simulations except I changed the transition time from 20 ecosystems to 50 to 100 to 200 and as you see as you um as you increase the transition time uh your your gaussians uh the intersection of the gaussians stays near the same but the width of the gaussians uh decreases and you get the values move closer to the actual delta g this this works decently well for the short transitions because we're going from a hydrophobic residue to a hydrophobic residue that are nearly the same size but for systems where we have very dissimilar residues oftentimes you do need long longer um transition times 100 or 200 pico seconds from long uh next we we select a different number of frames to analyze rather than using 100 frames uh as as displayed in the bottom here uh say we use 20 or 50 frames 20 or 50 transitions um you can see that using fewer transitions over uh fewer transitions increases the uncertainty but the estimated free energy remains similar overall so in this way you can you can tune the number of transitions in order to uh in order to reduce the error so now that we have this estimate we're going to use this estimate of negative 6.27 kilojoules per mole now that we have that estimate we can complete the thermodynamic cycle here is the thermodynamic cycle that Barrett showed earlier and as as he explained we can use some of these unfolded uh these unfolded uh mimics these gxg peptides where g is glycine and x is the amino acid of interest so we use that to simulate the unfolded state and we do that exactly the same as we did with this folded protein so when we add up all of our values uh from our simulation we would estimate that the phenylalanine cage the delta delta g for that mutation on the on the folding free energy is 14.3 kilojoules per mole and we actually do have an experimental value for that of 12.5 kilojoules per mole so we were happy to see that uh that there was good agreement with experiment in this case uh next i'm going to go over just a few considerations that you you might want to take into account if you want to use pmx first pmx uh does not use or does not do proline mutations uh because of the changes in the backbone topology second if you need charge mutations running a typical simulation would cause problems because there's a change in the net charge of the system over time and that will create some artifacts so we suggest a similar setup as Barrett uh had presented earlier where you have both the the folded and unfolded protein in the same box and you can read more about that in the paper that i have that i have cited below here and finally these terminally modified residues are not supported you can't um you can't mutate them with pmx and with that that is all that i have to present today and we would like to thank and acknowledge our great members especially uh Danielle and and Vitas um these two are the the main contributors to pmx and we would like to thank the audience for their attention and we welcome many questions thank you once thank you bet now we have already several questions and first we have a couple of questions questions from wow damas i hope i pronounced them correctly and i'm going to unmute you how can how can you hear us no okay i'm going to read out the question so the first question is is the unfolded wow type and mutated proteins in related the entire protein or just a portion around the mutation um right so maybe we went a bit fast there what we usually do for the unfolded state because it's it's we think it's it's a rather um broad ensemble that is difficult to to simulate in a converged way we simplify that by um simulating a short peptide and so we usually actually use a tripe peptide to model the unfolded state not even in the context of the sequence of interest but usually like a gxg tripe peptide so the the peptide of interested a flank by two glycines that also means of course that um there are also only there's a limited number of mutations that you can do in such a tripe peptide so we have them all tabulated and you can just um look them up in our tables you don't even need to simulate the unfolded state yourself if you don't want to thank you i will uh let actually how has two more questions but let's uh let's sweat uh vanguetti uh sweat can you hear us hello hi hi set yes we can hear you can ask your question oh so uh i was wondering is it possible to create topology files for disappearing residues instead of mutating them um yes um that is uh well not out of the box but it's actually um a rather um uh and similar exercise so you mean extending a protein chain on the end or the c-terminates right yeah either extending it or like disappearing at like removing the residue from yeah i mean it's it's um it's not something that you can do with the current software but it should be easy to add as a feature because it doesn't need any new functionality so if there is any interest in that then uh please drop us an email and and we'll put it on the to-do list sure thank you and i had another question so um what if you have to modify a residue instead of mutating it to one of the default residues is can pmx do that a phosphoserine or something like that yeah yeah um well then you would have to um dig a little bit deeper into the um um library files and if you download pmx you will see that um there are mutation libraries for all of the force field that we um support and um that would mean to go to your force field of interest and then for example if you want to mutate the serine into a phosphoserine um you just copy those building blocks and um use um yeah the analogy for example of what we have done for mutating a serine to a three-umeen um you use the same type of of um approach then to create a morph from a serine to a phosphoserine okay so that is well it's it's um it's not as easy as running a supported mutation for sure um but also there the pmx functionality will um should help you a long way so as long as i have all the atom types and correct structures i can just add and actually okay if you have no parameters for your modified amino acid then we cannot help you of course oh yeah that's right yeah i get that thank you okay thank you and uh question why you are on kills hello hi hi so i wonder uh you have the you intended to use gromax package but you didn't test any of the gromax force fields so there is a particular problem with this force fields for this type of application no it should be um um sort of um wait forward to also support gromax i mean we didn't have a uh a real urge ourselves to do the porting um um because in in the number of tests that we ran previously um we sort of came to the conclusion that we prefer a modern amber or charm um force fields for most purposes and so it was just a matter of priority that we didn't have around to supporting gromax um yet okay and another question is um so if i use the web server that you to generate a mutation for instance but if i want instead of uh calculating by uh affinity uh energy free energy for a folding protein but i want to calculate free energy for a protein ligand complex then i would upload the complex of the protein ligand uh and and generate a mutated let's say that i want to generate a mutation in the ligand yeah i i think that there there are two possible things that you might mean here so let me answer one by one the first one is what if i am for example i'm interested in the ligand affinity for a wild type protein versus a mutated protein then you can just use pmx as we presented it um and and if you want to use the web server then i would suggest uploading the protein without the ligand um let pmx do its thing and download the um the topology and the coordinates for the um uh with the mutation inside and then add the ligand afterwards and then you can go ahead and and you will get um the the difference in um ligand affinity due to the uh mutation if um if you if you want to mutate something on the ligand side um then uh this is something that is um um also in progress of being supported by pmx but we are still developing that at the moment so that's not something that you can run um out of the box as of yet okay yeah that was the second thing that was a what i have in mind similar to what you explained the first example you gave before go go into the 40 example you show this alchemical modification in the ligand right to calculate the free energy of magnesium yes because it's the frequent or a frequent example of how these thermodynamic cycles are are used in practice and that's certainly also something um uh we are um doing it's just that with the protein mutations in principle there's only 20 times 20 possible mutations so that's a whole lot easier than doing the 10 to the 60 square mutations that you can do in chemical space so that's why um uh we are taking a little bit longer there to reach the same kind of accuracy but just one more thing like if my ligand is a peptide and i want to mutate just for a regular amino acid could i upload the ligand alone in the web server or then it's a protein just like any other and you're ready to go okay thank you okay uh we have just a few minutes i'm going to read a few of the next questions sivastan is asking does pmx plan to support mutation of nucleic acids in the future um yes already in the in the very near future in fact so the the previous version of pmx actually supports nucleic acids it's um it's not supported now because we we went to newer force fields and also the web server but um so either if you um use a the previous version or the next version will also support nucleic acids again okay uh we have one question by musumi khazra he's asking whether the last slide the blue and red color bar represent two different states of protein system here probably um and no it's the same well both represent a transition between two states red here is the um is the are the so-called backward transitions where lambda is switched from zero to one uh from uh yeah from from one to zero sorry and blue goes from zero to one so um um both are coming from transition trajectories and they just are different in the direction of the transition thanks uh there is one question by salman serini uh he's asking whether it's it's possible to calculate the free and the combining of a minos to a metal surface in aqueous solution to a metal surface yes in in water solution um well we cannot help with the metal surface but if you have that setup then yes um PMX will generate the dual topology for your protein mutation and then you should be able to calculate the free energy the different differential binding to the to the metal surface one would only need to work out what is the proper thermodynamic cycle there so it's once associated to the surface and once dissociated maybe something like that but that all depends on the question you want to answer one more question from uh wow why gxg mimics a x a mimics would be the more would be more standard really um he doesn't have a microphone so i can't put him on okay yeah and i mean gxg there is not a real good reason to use that in um in uh when we were were um generating the software and and playing around with it and we we um simplified the unfolded state more and more and we came to the conclusion that actually the gxg works surprisingly well um there are probably also other choices that work um just as well or maybe even better it's just that sort of by accident we found that this works um surprisingly well and and and helped us to get these accuracies within 1k cal from from all from the experiment thank you berth uh this is a very interesting discussion there are a few other questions that could be followed up but unfortunately we are running out of time uh i would suggest to all the attendees to use the forums at ask.bioxcel.eu we have a category there for the free energy interest group and feel free to start up topics there we can follow up the discussion you can continue with the questions that you have today and also i would like to let you know that our next webinar will be on the 30th of june from uh the same time for p.m central european time and next webinar will be on qm and m approaches using cpmd specifically with application to biomolecular simulations this is another of the codes that bioxcel is uh working on will be presented by emiliano impoliti from new league and that's all for today i hope you will you have enjoyed today's webinar there will be a recording on the website so you can watch it later you can share with your colleagues and again keep in touch and if you have any questions please feel free to use our support forums on ask.bioxcel.eu or get in touch with us through any of the other channels thank you all thank you very thank you guys and we'll get in touch again bye okay bye bye


Well, I'm really pleased that the energy productivity of the U.S. economy continues to improve. When you look at GDP growth and energy consumption, there has clearly been a decoupling in that area. And as a result, we've had significant emissions reductions. In terms of changes to the U.S. electricity mix, you asked about renewable energy, a very exciting story. I mean, we started the decade at about 8 percent of generation coming from renewable energy in the United States, and we ended at 18 percent. So very big changes for a sector that people said, you know, really couldn't change in a decade or multiple decades. You know, the old saying was, you know, it's a hundred year industry and things take a very long time to change. Well, we've seen very significant changes in renewable energy in just a short period of time. And when you compare renewable energy at the end of 2019 to other segments of our electricity mix, we're nearly on par with nuclear. Nuclear represents 20 percent of the U.S. electricity mix and, you know, renewable is our mainstream. So the 2020 Factbook focuses a lot on this concept called the empowered consumer, and the empowered consumer can be at the household level or it could be at the company level. And in 2019, we saw 13.6 gigawatts of procurements by businesses for renewable energy in the United States. And this was a record-breaking year, and now companies are really driving demand for renewable energy. You know, at the household level, opportunities for rooftop solar or other distributed generation sources combined with energy efficiency technologies allows consumers at the household level to do things they really couldn't do before in terms of their energy choices. The Business Council for Sustainable Energy seeks a market-based economy-wide approach to addressing greenhouse gas emissions. We'd like it to be inclusive of the geographical resource mix of different parts of the country but have strong and aggressive goals to reduce our emissions. We need to follow what the scientific community has said we need to do in terms of lowering our emissions. And we feel that the results of the last decade, the growth in energy efficiency, renewable energy, clean generation sources like natural gas, give us a foundation to achieve those longer-term goals.


The person wasn't found for a couple weeks and the influence has been sitting in the home for 19 days. Some of his body was leaning against the door during the decomposition process, but it's actually a piece of the skin.


So, well, good morning everyone. I am Elena Perez Tirador, and I am here to introduce decentralized science project and how we are working towards decentralized peer reviewing. This is my first time talking at a conference and also my first time attending one, so well, everything is new to me. But I'm happy to be starting at this one surrounded by people willing to improve science by the means of decentralization. Well, this talk will be structured as follows. First, I will briefly introduce myself and the project. Then I will explain the research and design process we are following. And I will show a mock-up to illustrate how the current iteration design looks like. I will give a quick overview of the architecture we are working on. And finally, I will explain the current position of the project and what we are looking for right now. So the first half of the talk will be centered on the functionality of the application we are building. And then when I am talking about the architecture, I will explain how the decentralization comes to action. So, well, as I said, my name is Elena Perez. I am a recent computer science graduate and a senior mathematics student. And I joined decentralized science four months ago when the project started. And I work here as a blockchain developer. Regarding decentralized science, we are a team based in Spain building a tool to help with the processes in academic publishing. In particular, we are aiming to improve the process of peer reviewing. We aim to do this by increasing transparency and also by enhancing the process of finding reviewers. We, our target, is the growing open access publishing market and we are willing to work in creating an open ecosystem by extending existing tools. This project has received funding from the European Union's Horizon 2020 research and innovation program, in particular within the framework of the ledger project. Which if you are interested, if I am not mistaken, I think it is an open call right now and it is a grant for blockchain based projects. So maybe some of you are interested and you should look it up. Well, first, when the project started for the first months, we wanted to learn about the problem in order to build an actual solution that would help people involved. So the project started with the first research phase in which the first version of the first concept of the product was designed. We followed the directives of design thinking and lean design. So we will be continuously iterating different designs as further interviews with potential clients and pilots and all this kind of stuff is done. So by now we have done several interviews with different actors of the system, such as journals, conferences, associations, universities and reviewers. From these interviews we found out that there are several problems that are shared among all the entities we interviewed. These problems are first, finance suitable reviewers for paper that have the needed knowledge of the field. Second, once these reviewers are found, get them to accept to review the paper. And finally, once they have accepted, there are several possible outcomes. Some of the reviewers deliver on time, some of them do not, and some do not deliver at all. So taking all this into account, we started building our business model canvas to define aspects such as customer segments or a value proposition. So to solve these problems we have found, we define a value proposition composed by three main functionalities. First, a reviewer specialized search. This is when an editor is looking for reviewers to review a certain paper. This functionality will help find which ones are the best regarding both the content of the paper and the reviewers' interests. Then time or ability statistics. This is how many times each reviewer has delivered on time or has delivered late or hasn't accepted to deliver in all these situations. And finally, transparent peer reviewing. This is making review reports publicly accessible. Among other advantages, such as increasing transparency, this can be combined with the first functionality improving it. We are to implement these functionalities. We are integrating with existing platforms, so we reduce the friction for the new users. Well, regarding the peer reviewing and publication process as a whole, we come to action right after an editor has accepted a paper for review. So for this reason, we are not building a standalone application, but a tool that can adapt and integrate to existing systems, such as, let's say, OJS for journals or expert review as a plugin or extension. Now, I will show a mock-up to explain the functionalities we're currently working on. Since this is just the first situation in an InDesign process, this will not likely be how the final application will look like, but it serves as an example of what we're working on right now. First functionality is the search. As I explained before, after an editor has accepted a paper for review, they can search reviewers for a certain topic, also applying filters if so desired. So once the search is done, a list containing the reviewers is displayed. This list contains the name of the reviewers, the reviewing interests, and a list of the published papers and review reports in case they are public. Also, it shows the reliability statistics, which is the second functionality, and this is how many times they have delivered on time or late on all this stuff. And finally, as I just said, peer reviews are shown in case they are public. This is possible thanks to the third functionality, which was transparent peer reviews. Reviewers can make their review reports public if they so desire, thus increasing transparency. Now, I will give a quick overview of the architecture we're currently working on, as well as the architecture we plan to build as the project progresses. So here's a diagram showing our current architecture. We have an existing system, like, let's say, OGS, with its own centralized logic and storage. What we are building is an extension that accesses the system's centralized storage to gather the data. This is done via a GraphQL interface. Once this data is gathered, it is processed by our system, and then the processed data is displayed in the UI extension, which is integrated in the UI of the application itself. But well, this is still a centralized approach, and we're aiming for a wider structure. As shown here, once the base system is working, it will be easy to add the IPFS interfaces to access other decentralized storages, such as IPFS anti-blockchain. This decentralization brings several advantages. First, transparency. If all the information is public, journals and conferences will be easily able to show that their processes are fair and solid. Second, decentralized publication brings open access by design. If all the papers and the information is stored in a public and decentralized storage, it becomes harder to impose barriers to limit the access to the information. And finally, this way, we're also building an ecosystem of journals and conferences sharing information. What I mean by this is that a journal will not only be able to access its own list of known reviewers, but also a list shared among all the journals and conferences that are in our system. So this means more data and more valuable. And finally, since these interfaces we're building will be public, and because, well, the system will be implemented and it's open sourced, and also the storage is decentralized and public, any external client will be able to use all our interfaces to access the underlying system before enabling this being an enabler to building an open, decentralized ecosystem of peer-reviewing applications. So well, now to... Well, I forgot something. Oh, no, sorry. And now to close this talk, I will explain what's our current position and how you can help us. So as part of the agile development methodologies we're following, we're currently doing two pilots, one of them with the editorial unit of the Complutense University of Madrid and the other one with the Iberamia Association, which is the Iber-American Society of Computer Science, in particular for the Journal Intelligent Certificate. So if you are interested in our system and would like to try it, or maybe you know someone who could be interested, please contact us so we can see how we can help you. And also we are still open for interviews with different doctors in the system. So if you are or you know someone running a conference or a journal or an association with journals or conferences, or maybe you're a reviewer or something like this, you can also contact us and we will be happy to talk to you. So with this being said, you can find us in any of these platforms. Thank you very much for your attention. If there's any questions? Are there any questions? Yeah, great talk, by the way. Thank you. It was the first time, I can't believe it. Yeah, it was professional. Very good, yeah. Again, nice talk, thanks. One of the greatest challenges for editors is finding reviewers and building that pool and maintaining that pool and it turns over over time. And reviewers that an editor has used in the past will be busy. They could be reviewing other works and just don't have the capacity. Can you speak a little bit more about how you'll build that pool, how you'll identify these reviewers in ways that may complement or be different from how some of the conventional peer review systems that publishers use today are using? I'd be interested to understand how you're thinking about that. Yeah. In the interviews we have done so far, we discovered that most of the editors have their own lists in a spreadsheet or something like that. There's no specific system for them to store that, as far as I know. So our initial pool will be those like databases, manual databases these editors have with the review as they know. So that will be uploaded to the system and shared among all the journals. And then as far as the reviews are done, the review reports are public and the result of those reviews will also be public. So that will also feed the information we have about the reviewers. And I don't know if that answers. No, that answers thank you. A suggestion for you because there are other services that index the scholarly and scientific literature. And they've built products and systems to provide recommendations to editors to identify potential reviewers and such and to build their personal lists of relied upon reviewers. So I think a suggestion for you is to look into what some of those services, how they approach building that resource and think how you can at least match if not improve upon the recommendations that those systems are able to deliver. Thank you. Thank you very much for your very nice presentation. Very concise and up to the point. But my question is what a little bit troubles me about your project is that the problems that you identified about peer review are more or less familiar to people in academia. And there are already a lot of services and products which are basically the same functionality. I mean peer community and peer review, even pub loans, which essentially is just the, I mean it has a big competitive edge over any new kind of project. And also there's a blockchain-based project which is supported by Digital Science, if I'm not mistaken. It's called Blockchain for Peer Review, exactly, by Catalysis. Last year they had a presentation which met with another critical reception. But still, but they're doing basically this landscape. And my question is would be how do you position yourself in this landscape and how do you find a place for a product in light of the situation where there are many competing projects to improve peer review? Thank you. Yeah, I guess. It was a little what I tried to explain here. Since everything is open sourced and public and decentralized, we will kind of enable the existence of, let's say, a decentralized pub loans. Or yeah, pretty much we are like building the basis for any decentralized application related to peer reviewing to be built. We're trying to facilitate that. And yeah, I think that would be it. I don't really know. Thank you very much. OK.


Legal City Police Commissioner Bioduala B has clarified that residents who operate motorcycles for career services or other private dealings will not be affected by the latest Okada ban. The latest ban on Okadas in the state comes after a sound engineer, David Emo, was allegedly lynched to death by motorcycle riders in the leaky area of the state over a fair dispute. Police commissioners stressed that the enforcement of the ban this time will be more thorough as most Okada riders are criminals and must be taken off the road. When I've been joined in the studio by Ola Bordiogini, the councillor would F1 and also doubles as the deputy council leader Lagos Island East LCD. Thank you for joining us on the news this night. Good evening and thanks for having me. This is not the first time the government is actually banning commercial motorcycles on the highway but it seems the enforcement is not successful. What's the local government doing to ensure that this ban is implemented? Well, as a government, as a local government, which is the closest government to the people, what we are going to do or what we have been doing is to enlighten our people, to make them see reasons because the government is there to make laws and then there's no government that will sit back and watch its states or local government being turned to a lawless territory. So the government has made the law and the people are bound to obey the law. So we as a government, closest to the people, have been enlightening our people, letting them see reason why they have to respect the law because the law must be enforced. Now the recent damage to the co-bridge exposed some market problems in Lagos. How are laws enforced on street trading in Lagos? Well, there are laws banning street trading in Lagos. At the state level we have what we call the central business street tax force, the CBD tax force. And as a government that I represent that I serve in, in Lagos Island East LCDA, which equal bridge falls under our jurisdiction as well because we have two councils in Lagos Island. We have the Lagos Island local government and the Lagos Island East LCDA.  But you know, people do really like to obey the law, that is why the government are there. And then we have created, we have set up the, in our local government we have the environmental sanitation core. So their job basically is to go across all the roads, all the streets and markets and ensure they work in collaboration with the CBD and other law enforcement agents. Okay, so what are these laws that being enforced on street trading? There's a total ban on street trading, but you know, you cannot ban and at times it can be difficult when you ban people from trading and you are not providing alternatives for them to sell because naturally people want to survive. They want to look for a way to sell their wares and survive. And in Lagos, we have it on record based on statistics that over 10,000 people come into Lagos every day with the sole aim of living in Lagos. So we can imagine the influx of people coupled with the recent security unrest in the southeast part of the country. So what we are doing is to develop more markets and you know, Lagos Island local government as it is, does not really have much space for markets, but still the government is working particularly at the local government level to develop markets within the local government and these are markets that can be affordable, not price to eye for people, affordable markets for people. As I speak to you now, there is a project going on on Simpson Street in Lagos Island. Some blocks of shops have been developed and when they finish from there, the Zecliff chairman is going to develop another market at Olu Sura axis. So these are alternatives that we are providing so that we can get people out of the streets so that they can have somewhere to trade. All right, thank you very much. I need us to focus more on uteristiveness, especially in inner cities of Lagos. What's the government doing to get rid of street orchards in these areas? Like we all know, and there's no denying the fact that that has been the current issue in Lagos Island and the government has been doing its best. Every government that has come has been doing its best. And this government that is in place now that came into power July 2021 has been doing its best also to ensure that the menace of uteristiveness in Lagos Island is eradicated. You know, it is not always easy, it won't be easy for it to be totally eradicated, but at least it will be brought to the barest minimum, their activities. So we have a skilled Lagos Island East LCDA skilled acquisition center, which is being upgraded. As I speak to you about two months ago, some equipment were brought into the skilled acquisition center. Over 100 industrial sewing machines were brought in there by the executive chairman of Lagos Island East LCDA, and then some other equipment too were brought in. We have different sections there. We have fashion designing. We have soup making. We have barbein. We have tailoring. We have R&D engineering there. So we are trying to remove those guys from the streets. We are trying to bring them to, you know, it is very important that we give them what can keep them busy. So it ends the need for us to make sure that we train them. Okay, so I need to ask, these motorcycles that are being banned, wouldn't it affect state revenue in a way? State governments, you know, I'm a lawmaker at the local government level, and I'm very privy to the making of our budgets. And it is, I can tell you categorically that the state government and the local government, no government collect any revenue from Ocala riders. Because there's a ban on the activities. The first law was made in 2012 by the administration of Governor Bagile Fashella. And then it was, that law was reviewed in 2018 by the administration of Governor Akiyomi Ambodi, you understand? So there is a total ban on the activities across all the 492 roads, major routes in Lagos. So there's no way the government is going to affect the revenue of the government because in the first place, government is not even collecting revenue from them. If you see anybody collecting revenue from Ocala riders in Lagos, they are just doing that illegally. There is no body today in Lagos, no government, either at the state level or the local government level, that is collecting any revenue from Ocala riders. So finally, what then do you think is the solution? Because it has resulted in violence in the state current? Well, people are always, people are always, you know, want to resist enforcement of the law whenever a new law is being introduced. But that is where the security comes in, that is the security oppressive, that is where the activities comes in. Once the security oppressive are resolute, that they must enforce the law, then that law will stand. We have seen it work. It worked before doing the administration of Governor Bagile Fashella. So it all lies in the hands of our security oppressive. As a government, as a democratic government, we can only make laws, we cannot go to enforce. So when we make laws, we now pass it over to the security oppressive. It is not led for the security oppressive because even some of those security oppressive themselves too, they have bikes that they use for commercial purposes. So whenever they are determined that they want to make the law work, of course it will work and nobody can stop the government because the government is greater than any individual. Thank you very much for joining us on the news tonight. Thank you very much for having me. Hello, hope you enjoyed the news. Please do subscribe to our YouTube channel and don't forget to hit the notification button so you get notified about fresh news updates.


If you're young and you don't have a family, you should go live in a business entrepreneur house. 20 entrepreneurs live in there, on your level or a little bit ahead of you. Every time you're eating lunch, you're talking about some new idea. My best ideas weren't always my own. Getting on YouTube ads, I was out at dinner in Hollywood, but you execute it, but you have to execute. But you're already, you're not a procrastinator. So you're different.


Hey guys, it's Nate, aka the Foot Accountant. Welcome back to the channel. I sat down this evening to do some pre-weekend league rewards investing, and I thought of a few things that I wanted to share with you guys. It's kind of like a trading tips, kind of like a market talk, trading tips, analysis video, just looking at the market and some stuff that I know is today, and stuff that I've been seeing, and it's kind of making sense to me right now. Regarding Team of the Week cards and Scream cards, I want to talk about the rarity of cards this year, especially in terms of special cards that are in packs. Right now, when I'm recording this, it is late Wednesday night, and we just got a new Team of the Week impacts today on Wednesday. Last week's Team of the Week was pretty solid. A lot of people liked it with Ben Yetter, with Griezmann, Hummels, Ducore, 9-Golan, DeMaria, a lot of really solid players in that Team of the Week, and we saw a lot of those players rise. Even in a couple hours out of packs, you've seen 9-Golan go up a decent amount. We'll go over here to football and check him out. 9-Golan is 93,000 coins. This card just yesterday, I believe, a couple days ago. Sunday, he was 72K, and on the hourly graph, where was he at yesterday? He was 80K, and he's already up, basically 10K in 24 hours of being out of packs. Some of these other guys in Team of the Week 6, Ben Yetter's up like 40,000 coins. Griezmann's kind of chilling still in the same area. Hummels is up a couple K. Ducore is up like seven or 8,000 coins. So what is the deal with this Team of the Week, and why did this Team of the Week rise so quickly out of packs? Well, there's a couple things we have to keep in mind with this. First of all, the cards that rose, like we look at Ducore as well. This is just a meta card that a lot of people are going to want to use. Look, he's up 10,000 coins since his lowest point on Sunday of last week, and basically that's what I want to look at in this video is how do we know what cards are meta, how do we know which cards are gonna rise out of Team of the Week, and how much are they going to rise? This week really gave us an answer to that and how that's gonna work in the market this year, pertaining to pack SBCs. Even we had a promo running, so there were promo packs in the store that EA was refreshing every day, but we didn't get as many pack SBCs during this week of Ultimate Scream, compared to the first week of Ultimate Scream, which I think really affected the supply of these Team of the Week cards today, and I really, really do think that that played a huge difference into that today. You think about it this way, there were a couple days during the second, basically since this Team of the Week has been out, or the second week of Scream, we've only gotten probably four or five pack SBCs, unlike during the first week, when that first Team of the Week was out, we had two SBCs almost every single day, so every single day those Team of the Week cards and Team of the Week five were getting pummeled by pack supply. You guys probably remember why not all of them. People started looking at Thursday investing at around 150K.  This is him right here. So he was like 140, he went down below that, went to 144 on Sunday, and now he's rising back up after he's gotten more rare, he links to new Team of the Week cards, he's Liverpool, he's out of packs, but it took him longer because they had all the supply during the week. His load, we looked at the Team of the Week six cards. This cards, these cards from this past Team of the Week, a lot of their lowest times on the market were on Sunday. We just looked at Sunday, right here, 241,000 coins for Ben Yetter, boom, now he's 289, and of course they did just look at Ducoray and Nainggolan. Both of their lowest points were on Sunday, but if you go back again and look at this Wijnaldum, Sunday he was 170, and he went down to 149 by Wednesday, and that was because of all the SBCs that were dropped in that timeframe. That week, the Team of the Week five was in packs, we had under the spotlight, we had the UEFA Marquis matchups and the Champions Path UCL SBCs that came out during that week, and we were getting like two of the Scream SBCs per day. Like the Dracula's Army, the Tomb Raider, the Blackcats, Cursed the Werewolf, Beware the Lion and Zombies, we were getting like two of those SBCs per day when this Team of the Week was in packs, and that really, really affected the supply. Now, this week again, we didn't have as many SBCs for Team of the Week six when they were in packs, and that's why you see some of these cards rising out of packs already, and that's kind of the big thing that I wanted to make well known to you guys this week is since we had a decrease in the amount of those pack SBCs, going forward from here, if we see that again, or if we see a heavy amount of pack SBCs during a weekend or during a Monday or Tuesday when we're thinking about, hey, maybe some of these cards are gonna be good to invest in for Team of the Week, if there's been a lot of pack SBCs and the prices have done nothing but go down, that's something that we might wanna stay away from actually investing in because we know they're gonna have that much more supply. Think of that graph of white and all of them who usually, like this Ducorio was cheapest on Sunday, most of the times in a non promo week when there's not the promo packs, there's not a lot of stuff going on midweek, the cheapest day for informs is Saturday night or Sunday, but when we continue to have those pack SBCs, a lot of packs open during the week or league SBCs as well, that could affect the supply of a lot of these cards. This Team of the Week right here with Ducorio, that Team of the Week six, they escaped the initial Bundesliga League SBC frenzy, I believe, because I think that league SBC came out when, actually that came out right away. The Bundesliga League SBC came out right when these cards came into packs. Now I'm remembering, that is correct. If we go back and look at Ben Yetter's graph, the Bundesliga League SBC came out on that Wednesday and these cards were insanely low on that Wednesday because they had all those Bundesliga League SBC packs, other pack supply SBCs all being opened at once, first day and these were actually the cheapest on the first day, that's why you see Ben Yetter 1-9-7 all the way up to a 2-6-2, then down to 2-4-1, weekend league sell-off, and then bam, back now up to 2-8-9. So the league SBCs do play a big part into this as well. I really don't know if the current Team of the Week is low in terms of, I don't think a lot of people are doing the Chinese league SBC today. I could be wrong about that, but just by the way that these prices look, they are pretty low and they are pretty cheap. So maybe some people are doing that SBC, like a lot of these cards, if it discard already. I just don't know if these cards are gonna rise up and act like we had last week with Team of the Week 6 because there's just not as many meta cards in here that you would use. Bellarabi would be meta, Mbappe, Fabinho, and Kulu and Pulisic, is that really it? I mean to be honest, Saul is sort of meta but like nothing super-duper crazy, but this Team of the Week is something I'm gonna be watching this week, especially for a guy like Fabinho or Bellarabi for a lot more budget, informed card that you could use to spice up your team. A guy like Bellarabi is somebody you would wanna watch this week and watch to see if we do get that SBC content throughout the week to really see if he's gonna get a lot of supply, which might make his card a little bit less investable during the week. So let me check his price right now. Footbin says 51,000 coins, which is exactly where he's at. Three star week, four star skills, six foot. Not the best card ever, but it's fast, it's Bundesliga, it's a right mid, his direct competition is Serge Nabri, stuff like that. We'll look at some of those cards for investing later on in the week. But I just wanted to make this video today to kinda talk to you guys about, hey, when you see the pack supply and the pack SBCs, that's a really good way to gauge how rare the team of the week is going to be and how good it's going to be to invest in those cards when they do go out of packs. That was the main thing that I wanted to cover today. I wanna talk about screen cards as well because these cards are just honestly very rare and this is something that we can learn going forward in terms of what cards are gonna be good in like when we get team of knockout stage, when we get some of those cards and some of those promos, what kinda cards are gonna be good to invest in out of packs based on their pack weight. And from what I've seen with these screen cards so far, their pack weight is pretty stinking low. Let's try to get all the screen versions of Deli Ali in here. How many pages of screen Deli Ali do we have? He isn't back in packs yet. Obviously he was in team number one of Ultimate Scream. So his supply is gonna be less since he is out of packs. Right now in the PS market at this time, he only has nine pages. It is later at night. There's only three million live transfers. So maybe he has like 18 pages or 20 pages during the day. But 20 pages compared to some of the other informed cards in this game. I did a comparison a couple weeks back with Slothon's Scream card and Harry Kane's Inform card. Granted that week that Team of the Week five that Harry Kane was in got a lot of supply in terms of that Team of the Week. But these screen cards are just very rare and this is one way that you can kinda figure out how rare the screen cards are gonna be is by going up here in the promo pack section and clicking the options button down below to look at the pack odds. Less than 1% odds on getting one of these players in a 25,000 coin pack. That's a really good way to see the odds. But the best way to see odds is gonna be later on in the year when we have lightning rounds with 100Ks with 50K packs. Doing the pack probabilities on those types of packs especially we did have 50K packs. I think on like Monday or something of this week they did put 50K packs in the store at some point. But still I think the percentage for the screen cards was like 1% which is very, very low especially for a promo this early in the game when they don't usually run lightning rounds there's less packs opened when they don't actually have lightning rounds out. So that makes the cards more rare and with that probability percentage being so rare as well that's just made these screen cards very, very rare on the market. Now I know these cards are coming back to packs today on Halloween. Happy Halloween by the way. Hopefully you guys have some good costumes or whatever. But Bernard is down 200,000 coins. He is down to 200K. So he's down a little bit. A lot of these other screen cards are down as well and I really like these guys rising out of packs because the cards that EA have given us for this set they're just cool, they're fun, they have good upgrades and they're going to fit into a lot of people's teams. Romagnoli, Delialli, Hamas Rodriguez, even guy like Odomendi is going to be very high. You see a big price drop on him when he goes back into packs this weekend? That's a card I would be very interested in because his price has not fluctuated a lot. So far, same thing with Bernard if you see him at like 190 or below we'll see what happens this weekend with packs if they do bring out a lot of pack supply, pack SBCs or maybe their first lightning round FIFA 20, that would be something to watch. Laxalt is getting pretty cheap in packs now in signate, almost 800K. Gamero under 100. A lot of people want to buy it for me for 600K. I have a feeling he could hit that this weekend. Adama 322, Payet 79. So we're just going to have to watch a lot of these cards in the next, this weekend basically because these cards do have a very low pack weight but if you see any sort of like sell off or any sort of panic on the market that's going to be your cue to pick up a lot of these cards. Even Bernard being down like 10,000 coins is honestly not too bad. I know some people that are just thinking, hey, these cards are down a little bit now. I know they might drop a little bit when they come back into packs but at least at the moment they feel like they want to take the chance go get a card for their team because they almost don't feel like it's going to be low enough because these cards are so rare. I still think the cards are going to drop at some point this weekend. I think EA has a pretty decently packed field of content a weekend filled of content ahead for us. So that's what I wanted to kind of say about that. I do feel like they could drop some more this weekend especially like guys that I already feel are very inflated like Adama Treore. I think he's very inflated but I do think these screen cards are going to rise a decent amount out of packs especially as we continue to get special cards in the game, continue to get cool cards that people want to use in foot and want to mess around with and try out. So Hamas Rodriguez is 322K with a hunter chem style. So still that's a card I think a lot of people are going to want to use and you can kind of, you know the informs and the screen cards kind of work similarly. The special cards all work the same at some way and form in this game because they all go out of packs at some point. Yes, they could return back in packs for whatever reason EA would do that but at some point they're in team of the week or they're in a promo week and they go out of packs after that and a lot of people invest in players to rise out of packs, which is a very common thing but I wanted to kind of bring you this guy bring you guys this video today talking about rarity of informs talking about stuff on the market in terms of when cards are rare how do you know if they're rare and how much pack supply affects rarity. I wanted to bring this video to you guys today talking about this because it's going to help us with our team of the week investing going forward and especially I'm eyeing some of these players in this week's team of the week. Again, team of the week seven, I mentioned Fabinho, I mentioned Bellarabi, I mentioned Mbappe, Saul and honestly in Kulu as well. It's not a bad card, not a terrible card. Pulisic maybe as well. By the way, Pulisic getting a dynamic image on the bench that's pretty rare. A dynamic image for a bench player is pretty rare but again, this week might not have as many meta players as one of the past team of the weeks did like with Griezmann, Ben Yetter and some of those guys in like last week's team of the week but there are still some notables in here that I think if we do have another week of kind of low pack supply and not very many pack SBCs, pack supply SBCs we could see some of these guys rise out of packs a decent amount. Again, so if we don't see a lot of those SBCs it's really kind of a tough situation because you're almost put in a position where you have to wait until Tuesday to buy like the day before they go out of packs is honestly when you have to wait to buy for these cards because you could buy on Sunday night but then you might get screwed by a pack supply on Monday or Tuesday, which then is an issue. So again, for these cards I would probably wait until Tuesday just to be safe. They might be up maybe two or three K from where they were on Sunday night like last week's team of the week if we didn't get supply or if we don't get supply that's where they could be again but that's kind of the safe route to do it. So I just wanted to bring this again to kind of inform you about supply, how to know when cards are rare and how to just judge the supply of cards on the market based on their pack weight value and based on the SBCs that we do get during the week. Now of course, sometimes crazy amounts of demand trump things like a Venetar card his card probably still would have rose out of packs even if he would have gotten packed a bunch like team of the week five did but team of the week six didn't get packed a ton and that's why we're seeing it rise right now in FIFA Ultimate Team. If you enjoyed this video, smash a thumbs up on it. If you have any questions, comment down below and of course, subscribe to the channel if you are new. It's been Nate, the Foot Accountant. Catch you guys later. Peace out.


Hey everybody Good afternoon, and thank you for listening to easy sex session for the poster saving bank of China embrace Mobile Internet by using open stack. So this is our easy stack VP for the solution Zhou Chong Yi and this is Jun Chen for the product manager and let's begin for our session in our Last one, so thanks everyone for hearing this. Thanks. Okay Good afternoon Ladies and gentlemen, we are very pleased to join the oversteck case study We are about to share the topic with the poster savings bank of China embraces mobile internet by using over stack my colleague Zheng Cheng and I Where as? Where be the speakers for this topic? We all come from the Chinese Homestack company is that My name is Zhou Chong Yi and I'm the VP for solution is that and the Zhen Chen is responsible for product design and development Today we wanted to share four parts about poster savings banks of China Also as a long PSPC first, I wanted to share some information about the PSPC Second I wanted to analyze Traditional bankings business purchase in this time finally my colleague where share some keynotes about financial cloud solution based on over stack and Why they choose is that Okay post savings bank of China PSPC is The fifth largest bank in China It was original department of China Post as The business close China post separate this this department to build PSPC Which is youngest big bank in China PSPC business Is very different because it's customer mainly come from country not city There are almost the 40,000 department in China and the nearby 490 million people's server server percent of the global people has become customer the asset of PSPC ranked 3rd in the global bank ranking of the top thousand which is named by the banker London based magazine in the past September PSPC has completed its IPO and listed in Hong Kong with the market value about 100 billion Hong Kong dollars This nearly the world's largest IPO this year As we can see in this table This is the total asset of China top 6 Bank banks and the PSPC is number 5 PSPC is the giant banks giant bank in China even in in the world with 7 trillion assets As some people may know the giant banks Include PSPC have a long road to to go in the mobile internet era Actually, they are suffered by internet company What are they worried about? There is a book called how Google works Which is referred to if the cost curve of the main factor of a certain Industry decline this industry will be changed today the cost of the users Interaction with with banks Can be extremely reduced by internet people can I'm sorry People can get the information and the finish dealing Without going to a real estate They only need to do is using their Mobile phones and to to make connection with the internet And the business include Connection information acquisition and transaction At this time the internet companies found some Better resolution to make the user still More comfortable on the internet without going to the bank and there will not be any advantage to traditional banks by the site location to serve the core customers as business model, but There is more advantage to Internet company which do business on the internet So they launched a battle to the traditional banks for three in three Areas first mobile payment, which is using violence to attract deposits second computation for small and micro loans market serve Interactive online banking to reduce the traditional bank middle business income So this is sense of the computation between internet companies and the traditional banks is to change business Change business model to serve the majority of people is that all? Core customer customers But the traditional bank find that they don't really understand what the majority really need as a result They got high cost and low profit When as they using traditional business model There are lack of the differentiation and attraction for their products at the same time the Internet companies has launched a series of Competitive products such as Ali pay you a bow web chat web chat the red package Which I checked the majority transfer their money from the banks to the Internet company account more even more more and more elite customers are also be our also be attacked also Sorry are also effective by the by this interesting products traditional banking continue to be eroded by the of course they Were not set without any fighting Involved using internet financial risk control model providing financial and non-financial service with through the through the plan for strategy and make promotion for small and the media user experience and Using big data and so on the main purpose of this innovation is To get more customer from the competitors There's there is There are some question be answered must be answered How to add users new users how to retain users how to improve the users activity how to improve the income of a single customer and how to control the risk when we solve this this problems we can attract more users and Scare out our business No matter we are an Internet company or Traditional bank That's the reason why we saw a lot of Financial groups and the universal financial groups emerged Driven by this business need cloud computing has play a new Role to the business we can see three at least I can see there are at least the three cloud computing requirement in Internet financial business first mobile direct sales the business model relies on Internet channel not physical side The IT challenge is a difficulty to predict the peak of the business Second interaction online banking based on customer interaction behave enhance users experience provide Real-time personalized the marketing capabilities This need big data outlets third production and the financial Collaboration provide in funding find financial products and the third-party services That means complex product and customer management This requirement are difficult to be achieved in the traditional architecture If we do so the cost must be high but by using cloud computing you can get the Advantage of cost reduction rapid delivery and the flexible deployment Today Opstack has been the Has been the number one choice for banks in China which Which one the tool which one to make their IT transformation such as ICBC IBC PSPC and so on Next my colleague Zhen Chen will introduce the PSPC's architecture. Please. Thank you, Zhong Yi Here we are the financial cloud solution based on OpenStack for PSPC so already the Zhong Yi has talked about the challenge the banking background and we will talk about the business challenge first the online Internet Payment and that the user between issues is become the most important issue about Banking in China, so they want to change themselves transfer the local traditional IT solutions to the cloud to support them in the Innovation solutions, so that's the business challenge background and why they choose cloud because They use easy yes cloud easy study yes cloud solutions to manage their data center servers to support the self-service banking the mobile bank and the internet loan the business for the next generation of the PSPC of this Background and we can see there was 63 and 150 and 650 Servers will be deployed in this year We are busy busy with these things to like the business come come online first as soon as possible and to the customer benefit we have the Use cloud you can have the innovation ability Because in yes cloud we have the ability to manage the container service. We have the database server service We have the big data all of these things is based on OpenStack, so yes cloud could could have customers to save costs and Same time customer can continue to innovate with the open and controllable technology Now this is the architecture design. You can see there are five Main component and there are four layer of this we can see in the base level There are eyes we can have the one where we can have the machine We can have the bare metal we can support one way and we have multiple network solutions we have object storage Nuts storage and the block storage and we have some policy service QS service All these things is to support the The ice infrastructure is stable and secure Beyond of that there are past solutions. We have the hard to big data cluster based on the Based on Sahara we have the web logic the traditional application Build on the OpenStack. We have the database servers. We have hybrid cloud service Using easy stack yes connector product. We have the container service now in the Previous session we have introduced our unified management portal to manage a Kubernetes cluster on OpenStack with the tenant-aware bare metal cluster and we have object storage. We have file service All this can support different type of business of the banking to do their innovation things and above that you have the micro service chain and cluster service which can manage for different apartment of the banking and Beyond that there are applications and look on the The right side you can see the cloud security and the monitor of the things because security and stable is the most Important things for the banking business This is the Function design you can see we have the Core service which based on no one new transgender and so on component of the OpenStack And we have do the this include the some mature and immature Component to the ESL solutions, which can Support different type of business on them and we have adventure service. You have billing We have tickets. We have upgrade. We have alarms Even management and the operation slot all these things you user can see on the white portal and we have monitor Solutions which can cover the physical and the virtual resources security related we have Combine with the AD LDAP and so on Authorization system and we have SSL in prediction all these things is to Make sure that the the ice cloud is secure ready and So for portal is based on the OpenStack So all the API is provided upon Banking have bank have their own portal or something can Direct T Cal the API to do their things This is a network architecture One I want to highlight the there are three separate network The DMZ the China private and the corporate network. This is the the PSPC requirement and we can adjust our Deployment architecture or line with the customer's requirement This is the highlight point because HAA is always the thing we want to talk about We first we support the live migration This is the if you have a one server Want to maintain you can live migrate your application to the other servers and the the most important is if you form a Host is that but there is a application on it. We have the host high availability Availability to make sure that the application is transferred to the alive host and there are 600 more than 600 knows architecture design you can see we have Three my circle cluster and the two MongoDB cluster. We have file controller to control this six 600 knows and There are two Rabbit IQ cluster which can separate the management Dataflow so there are four maybe zoom to Support different business requirement now, let's talk about why is it like because of All this I talk about is the things we already done, but we want to go further This is the is it like strengths. We have the leadership in China we have we are the OpenStack Gold members we had a Linux foundation several members and we found the open-stack China community and Mystery of the cloud computing opens your industrial lines member units and we deliver our service and the Product to state grade post saving bank China Union pay Industrial banks as express China telecom China mobile the novel Xinghua University and So so many customers all of this customer is There are six of the of the of them is Open-Stack community already mentioned there are eight Biggest customers in China we serve six of them And the second that we have more development strengths we have do the TC approved companies and New Gen TC approved companies to the global number 10 and we have the continue to contribute to the Open-Stack and Beside Open-Stack we have already done and continue to do the Linux foundation contribution and the safe community contribution And the for creativity creativity, we have the industrial dedicated cloud Yes, cast product line and we have the gardener 2016 cool wonder This is why we can do this because we have the engineer to do this There are top opens like an engineer in China. You can see the list There are many many familiar names and the Chinese name to do these things and we can We can continue to contribute the commies at the 1,000 and the 879 and the lines is 87,000 So there are two cores of the easy stack and we will do that do this In next year make it double or triple. That's what we want to do This is the easy stack contribution in past because we have already done the the ice infrastructure cloud service, so we want to build a Better pass platform, so you can see we have the 12 Megan Manila Sahara Morano and the Zaker all this we have the the the ranking is One side away, but we contribute this and we put it back to our Product and make it product ready as I said before a Side of the open stack we have do the Linux kernel the visualization storage network System quality and system tools you can see the Alex see the zoom safe The external all these things we have already do the the the code things to the community So make sure that our open stack deployment is stable and This this is for our requirement for our engineer team So if you want to compute to compute things you have to know Noah architecture kernel Libre Q mu KVM So you want to do storage you have to know sender you have to set and So on so you can see if you want to do open stacks better You have to know not only open stack things Is it like one to support customer innovation? This is why we do the things and the do innovation platform, so we have yes cloud yes connector yes cast and Some storage product line to support customer innovation So we have dedicated reliable and manageable is a stack enterprise product to provide a high quality is a stack sporting service and We want to create Open-stack ecosystem with the corporation in China to Retain a win-win and the open strategy and this You can take a picture. This is easy sex success story part of them and you can see The post-saving bank of China the China Union pay China telecom China mobile state grade This is just a part of it and we have supporting much more bank and financial things University and the state grade the water resource Different industry We have different solutions to support them to do the innovation things Transformation of their business and so on Okay, so this is all I want to talk about the the Open stack things and the easy stack do the post-saving bank Story, thank you. Any questions? so The the security is the base basic thing off we won't talk about but first now The isolation this is the first we can do to to make the security the server controllable and if you cannot do the isolation we can use many layer Secure functions to make the new business Secure because we have the the network layer. We have storage. We can separate the different user data to different pools we can do the Network security rules on the server on the controller and on the top of it we have we can use the the the Unified user management to control the access we have SSL and What's more we can integrate different SDN Corporation to use the the the high-level on the deep-level Secure functions to make the the network square. I That's that's Okay, anything else Okay, now let's finish here. Thank you for all listening for the sessions