Okay, welcome everyone to this workshop webinar for the BioExcel workshop in best practices in QMM simulation by Microsoft Systems. I won't take too much time, especially because people who have been to the previous webinars will already know what this is all about. Just to restate very briefly that what we're trying to do with this workshop is to really look at a variety of perspectives from very experienced practitioners who are using QMM simulation for bioelectric systems and look at what insights it gained about what could practice robust protocols for simulation, things to look out for, it holds, and also perhaps in some cases, some ideas about software to use. So to introduce today's speaker, we have Professor Maria Adriano-Ramos from the University of Porto. Maria heads the group of theoretical and computational biochemistry at the University of Porto. She has an extremely long list of publications that hasn't had a long list of high-profile propositions which I didn't want to put here, but I just want to highlight Maria's research interests and expertise, especially as relevant to the workshop. So Maria's research expertise is with regards to computational and symmetric catalysis, and there I thought what stood out to me in particular was a number of publications that she and her group have done on benchmarking and suitability evaluation of DFT functions, which I think is a really interesting topic and really valuable for people who are coming to QMM simulation, fresh, and I think she might have touched on this today in her presentation. Other areas that Maria has done research in and published extensively about dynamics, computational and geneticists, I think using a PDSA and my theatre blocking and director's covering. And Maria was also awarded in 2019 the Medina Vita, I learned so surprised by the Spanish role of science in the chemistry. I did my best. Okay, so I will, with that having said that, I will hand over the reins now to Maria. I just wanted to say hello to everybody and thank also Arno and Emiliano and Perit for organizing this series of seminars and obviously for inviting me to be here and to talk to you about studies on enzyme catalyzed reactions, which is quite a general name and today I am going to focus on mechanisms of enzymatic reactions and for that I would like to tell you first what I want to know from when we establish a mechanism of enzymatic reaction and to just give you an example of exactly what we want, my first slide features the phyldisophyte exchange mechanism, which is not terribly complicated, it consists on a nucleophilic attack by a methyl phthalate on a disulfide bond, it breaks it and another one is made. And one of my ex PhD students, Ruy Nevesh, who is now a, has a research fellowship in my group of research and has done a very nice work on the reduction of glutathione disulfide by PDI and using exactly this exchange mechanism, which is actually used in many systems in biology and what he did was what we knew at the end of it and all the other many enzymatic mechanisms that we have established is the, all the geometries and the energies of all the states that are important, such as reactants, transition states, intermediaries, products and by knowing all this we can actually visualise the mechanism, we know the steps, we know everything and Ruy has done a very nice video of which is done with all these geometries that he calculated. So this is PDI and here is the active centre and as you can see we've got for the first step of the mechanism we've got here the disulfide bond, the methyl, a phthalate that is attacking it, the bond is broken, the other one is formed and here it goes again, there's another methyl phthalate that is forming here, which is going to attack again the disulfide bond and form a new one. And as you can see, and I can't, I mean I was very happy to be able to visualise this, this gives us ideas and it's clear, very clear and that's what we want, we want at the end of the day to understand exactly what goes on with an enzyme when it reacts, yes the orders of magnitude of enzymatic rates of reaction based on experiment, so you can make an educated guess and that's what we use to do all the time and by educated guess I mean you know that if the rate of a reaction gives you a delta G of activation which goes say 30 kcal per mole or something is wrong, you've made a mistake somewhere, either the mechanism is wrong or the value calculated is wrong or the Hamiltonian is not good enough and provides that because with 30 kcal per mole the rate of reaction is too slow for any reaction to occur in your body and if it is a very important one you're more likely dead by the time it would sort of react with the delta G of 30 odd, so that's what I mean by an educated guess. So in 2015 and then it went on until 2020 we actually decided to do a computational study totally based on experiment and we went to the literature, we got out all the values of the rates of constant, experimental rates of constant, yeah and the rates of catalysis and basically what we did was we put it all together and here is and we just published that in 2020 and if you look at it and you've got here the percentage of occurrences in the individual classes as a function of the delta G of activation, so the stepwise graph is a sum of all classes and you have and the individual ones are with colors by color, so for example we started in 2015 with hydrolysis, it's the darkish blue and you can see that the dark blue itself goes from 12 kcal per mole to 22 kcal per mole but already 80% of that is in between these kcal per mole here, these numbers here and if you sum the whole lot, I mean you never have I'm saying from the values and we got about, so our study focused on a thousand odd experimental rates that were published in the literature and it very little time goes above 24-25 kcal per mole so that's what I mean if something is 30 or something is not quite right, so this is another way of you to know whether you've got the correct energy of activation and if you've got the correct energy of activation in principle you do have correct mechanism I'm not saying it's a hundred percent certain but you know you've got a pretty good chance of having it, so which are the the main problems that we were faced with during these years of work, first of all is the Hamiltonian then the long-range interactions then the reactional space and finally the conformational space, so this is a recurrent slide which is going to turn up quite often in the presentation because what I'm going to do is I'm going to focus on each one of these these major problems and tell you how we solve them, these are in fact problems that we have been dealing with forever not forever but anyway in 2008 for example we already talked about this and now we are in 2020 but we now have far more answers than we did then so now then they were real problems from the point of view that we didn't have any answers now at least we've got some answers, so with the Hamiltonian so which one should we use because there are many to choose from these are just a few CCSTD, LDNO, CCSTD, MP2, DFT, DFTB, semi empirical many more but one thing that you have to and there are protocols as well lots of protocols software to choose from as well but what we'll have to keep in mind is that whichever you choose it has to successfully deal with the nature of all energy contributions and of course which one to choose well that also very much depends on other things such as the group you start working with which will have their own expertise concerning a particular software usually a particular method as well or you know the computational power that you've got or or else even the software where you run your calculation sometimes you have access to some but not other so it very much depends on that but this is the main thing is that the Hamiltonian has to successfully deal with the nature of all energy contributions and if you're dealing with enzymes you have all sorts of energy contributions in your hands to deal with so I'll go a little bit more now with the Hamiltonian before passing on to other problems so the methods that we favor are when I say bioinformatics I put in here everything that sometimes we've got to use as well to help us setting up the system such as if the substrate is not docked into the active center we've got to use molecular docking if we don't know part of the enzyme or even the whole enzyme we've got to model it and do homology modelling etc and then we use molecular mechanics and quantum mechanics within the quantum mechanics we favor definitely DFT that's what we favor and but we also do mp2 ccsbt and even others but these are the main ones that we use we use QMM and have been doing so for many years and we now do QMM MD some of it anyway sometimes with QMM we have been running on Gaussian using onion and with QMM MD we have been using orca or cp2k mostly because they are software that are available where we can run our calculations and so we can do it without any problems and sometimes we have some problems into in setting up other software so for us DFT is the only practical QM theoretical level at the moment and and this obviously depends on the computational power that we've got the systems that we deal with however as you know functional performance is case dependent and as Arno was talking about just earlier on we started by benchmarking the DFT functionals before running our QMM calculations and we found out that in fact with some systems not all of them but with some the difference in the results was quite remarkable so we started dabbling with different functionals when running our or when establishing our mechanisms so so basically what we do is that we cut this is to benchmark the the functionals we calculate a reference potential energy surface at the ccsbt cds cds complete basic set level and then we benchmark the density functionals now this ccsbt cbs level has got to be has got to be done because when you benchmark a functional you have to benchmark or whatever you benchmark anyway you have to benchmark against something and so what we do is we calculate the something in ccsbt and we calculate the mechanism the whole mechanism with this small system that includes the zero content energy thermal effects the reactants and products of the term fluid I actually conclude I should do the same thing as you do the the enzymes as well and we then calculate single point energy values of all relevant structures that were calculated from this small system at the approximated level ccsbt cbs having geometry optimized with the three lip and you have then your reference to evaluate the accuracy of the density functionals so now you go to your whichever density functionals you want to look into and sorry I forgot to say this so but basically as a summary we fully optimize the geometry with bit three lip and then we improve the energy with the benchmark functional so when we do this we get accuracy which is close to the ccsbt cbs level in the chemistry which is the important part but it's not just the important part but obviously this is important and so you get a functional that is that mimics very well the or is very close so that's what I should say to the ccsbt cbs level and with a computational cost the dft triple the z level and a system size and cpu time scaling at the dft level which is excellent so this is our experience and so since then when we triggered that we have been benchmarking our density functions if you want to follow something like that you can use this first reference and in fact I forgot to put another one which is quite important which was a study which was done by Huy Nefs again sorry I've got it here and I forgot to pass it there which is from 2014 so it's earlier sorry the latest stage and this one was done and I forgot to put there the reference but anyway you can look it up and see this one which is basically the same sort of thing so here are the examples we did this so we've been doing them for many enzymes but these are four which then follow on to another example so that's why I chose these four and and these are the small systems that I'm talking to you about now if you focus on this one the time files and exchange mechanism it's the same one that I presented to begin with so that you would recognize all the selfish so what Huy did was that he set up this small system which mimics what happened in the active center of the enzyme if you remember the video that I showed with all the all the disulfide bonds forming and breaking and he set up the mechanism for this small system he also published here is I was looking it up but here is the reference it's JCTC 4842 in 2014 and then with this functional he then went on and did the enzymatic mechanism so that's what we do and nowadays it's actually recently we found out something which is quite exciting which is benchmarking at the DLPNO CCSTT level and if we look again at the same four examples that I just gave you a Pietro Piver which the student of ours has evaluated the performance of this method which has been which we owe to Frank Neese and his co-workers and at the same reference potential energy surfaces that we've been I mean we calculated these very accurate reference systems yeah so which are these four and others but Pietro only Pietro 5 only focused on these four because they are they they they they lengthy these things and so we've evaluated the performance we were not the only ones they have been established by Frank Neese as well and in fact but for these four this particular four which is our experience the DLPNO and so basically what was I what I was saying is that the results were very exciting from the point of view that we got a 0.5 kcal from all we were a difference from the reference ones the reference ones being the ones at CCSTT level and just two things that I was saying we didn't use any transitional mentals in this reaction just basically because they didn't have any transitional methods and they were the systems and the study and and we noticed that this is base dependent so you really have to use a good damning basis so this is more or less what I wanted to say about the benchmarking of density functionals and the Hamiltonian so what we obviously used the Hamilton the benchmarking to improve our Hamiltonian as best as we possibly can and software method delivered systematically better results than any density functional okay good let's go so this is what I basically had to tell you about the Hamiltonian or how we deal with the with that and how we improve the Hamiltonian that we choose and so we can carry on now and go to the long-range interactions now how important our long-range interactions we know that they are challenging to calculate technically speaking which model should we choose to use or I mean by we I'm talking about ourselves obviously I can only give my experience and or I I would only like to give my experience or should we just ignore them so what I can tell you is that in in an enzyme the long-range interactions are very many and they are they cover a very long range they go from 7 to 20 or can go from 7 to 20 angstrom so we have to be careful with them and I still just I don't have much time so I shall refer once study which focus on beta galactosidase which is an enzyme that catalyzes hydrolysis hydrolysis reaction of lactose into single sugars and as you see here so this is what really matters to me at this moment the yellow is the delta G of activation and this is a the active center of the enzyme this is a cut of the enzyme and this is another one so when we had a very small oops when we had a very small model of the enzyme with just obviously two small 35 atoms in the qm part we got extremely high delta G's of activation uh we then went on to 227 atoms in the qm part and sorry in the in the in the um yeah it was the qm part no this was a qm model which in total had 227 atoms so we cut the models so basically we used the cluster model and we also got a very high um delta G of activation but when we use a large part of the enzyme it wasn't the the complete enzyme but it was a large part of the enzyme then yes we got 15 kcal per mole always the same mechanism that was studied and which was was okay with the experimental result which is also 50 kcal per mole so in this particular case these long range interactions were determinant to get a good a good value for the delta G of activation which helped us setting up the correct mechanism um is this and there were no conformational changes here uh these these were really just long range interactions without any conformational changes so is it always like this no it's not so there are cases many cases in which you don't have such um such important long range interactions and they sort of um cut off much nearer the active center but you don't really know in advance when that is so so you have to be prepared uh for the long range interactions uh to be uh quite important for your calculation so we tend to use if we can the whole enzyme or at least the whole monomer if it is a case of uh many um of a dimer or trimer or whatever um but we we we are careful with the long range interactions we think they are important and we want to include them if we can um so for the reactional space well there can be great diversity of the reactional space with often quite unexpected chemistry when compared to solution chemistry so this means that you're in poor problems with the reactional space from the point of view that if it is complicated reaction your you have to uh sample this reactional space very well so you set up your hypothesis mechanistic hypothesis which can come from experiment or not uh and uh but whether it comes or not you really have to study all the reactional space which can be complicated and i'll just would like to uh run you through an example which sort of uh uh probably scarred my uh one of my very talented phd students build a fighter which very recently established the catalytic mechanism of human aldehyde oxidase and the whole thing's a bit of a nightmare because as you see the reactional space is very diverse it had two molybdenum atoms and they kept changing oxidation state uh quite a lot of steps of uh uh mechanistic steps and uh and it took a long time so um my advice if i can uh sort of give to uh each of the people that are not so used to doing this every day um the reactional space can be very complex and you really have to think that the more complex the or Hamiltonian the more difficulty becomes to solve the problem so you have to compromise uh you can't use a Hamiltonian that you know that probably we could give you a wrong uh answer but you have to compromise and still get one that will be able to give you the correct answer and also um establishing for me establishing an enzymatic mechanism is like solving a puzzle to a certain extent i every single bit of correct experimental or otherwise the information must fit and cannot be left out so you can't just say oh um this experiment or this experiment or this experience uh gives gives us i don't know the pka of histidine which means that it is um solvated or not or uh uh uh or not or whatever uh you can't just say oh i will disregard this no you can you have uh to to to fit it in and uh and nothing should be left out everything has got to to fit and so that you know that you really have got the the right uh mechanism and finally the uh conformational space uh which how determinant easy to sample enzyme conformations uh so a lot of people do q m m m m t kappa inelo metatynamics and all this and they do uh excellent work i mean really first class work uh other people don't work kappa inelo or anything else that includes dynamics this is not don't don't take me wrong uh is that i we have been doing it basically because we couldn't afford for a long time we couldn't afford doing other than q m m m and uh and we find that still do uh that we get a lot out of it a lot of detail out of it and i'll give you an example so that you understand my point of view and i hope you do um so let's start with single confirmation uh calculations so i'm talking about q m m m in which you start with a confirmation to start studying your uh your mechanism your your reaction mechanism and with it we can identify structural features of reactant conformations and understand very well the structure activity relationship uh so uh let me uh present to you alpha annuase uh this is what the enzyme looks like this is the active center uh uh we've got the sugar for substrate and two catalytic uh or two mechanics sorry uh two residues that are catalytic uh so we've got this glycosidic oxygen that is um sorry here that is protonated um by uh glue uh 233 and what happens is that this bond breaks and then there is a nucleophilic uh addition by uh 1 9 6 so it's not terribly complicated we established the mechanism for um the enzymatic mechanism we used onion so we did single uh confirmation uh calculations just one started with one confirmation that was basically the x-ray one um and uh we still had to optimize because of substrate to be put in place and all that we include we used um so p3 lip for uh the uh the geometry optimization and then m 06 2x for the energy um so we basically benchmark uh we included we did everything so included cpe so thermal effects etc etc and um um but we started with just one confirmation so you know the the doubt always uh is uh is there thinking uh what if i used another confirmation to begin with and to take away that uh problem we decided to run uh a dynamic simulation along one and selected snapshots from that md simulation uh these snapshots were not selected at random because you have to understand that when we run uh a long dynamic simulation the space the conformational space that you sample is incredibly large there is absolutely no way that you can sample or they can you can study with detail all these um all these uh different conformations so we wanted to sample those that were very near uh the the um uh the start of the catalytic mechanism because those are the ones that are going to be important for catalysis which is basically what we want uh start you know study catalysis but uh the catalytic reaction but with a different uh confirmation so um these these uh conformations that we looked for were those that were predicted to be adequate uh that uh that uh sort of uh uh were um followed adequate criteria for reactivity and how did we do that uh so we looked at our at our active center i included here well here are the catalytic uh residues i included this one as well last 300 because it's also it's not catalytic it doesn't go into catalysis but it's a structural um a structural uh residue because holds the sugar in place because of doing uh hydrogen bonding uh bonds with um the hydroxyls that are uh on these two carbons so what we did was we looked for the conformations that uh first of all the distance from arse 196 to carbon c1 was under uh 3.5 uh and strongly so i kept here the mechanism so that you are you remember it and then we also uh at the same time these conformations had to um um had the structural hydrogen bonds so these hydrogen bonds between arse 300 and the hydroxyl groups attached to c2 and c3 and the 2.5 angstrom uh so that's what we looked for those conformations and we selected 40 of them uh and we for each one of them we solved the mechanism yet again so we ended up with 40 free energy barriers of activation for each of them we repeated the exact protocol that we have followed for the first uh single conformation uh the x-ray basically optimized uh that we used to begin with and this is what we got so we got what we called a multiple pairs uh analysis so to speak in which you can plot this the activation barrier as a function of time and a function of each of the uh initial structures that you start with um to establish uh again and again again your mechanism and for the x-ray so the experimental value is 14 uh the x-ray we got 11 and look at this we have a very wide range of of barriers of activation and you have to remember that these were all chosen as being catalytic or near catalytic uh uh structures or structures good for catalysis so to speak with the distances good for catalysis and uh and these are the only uh in 40 of them uh the only four that we managed to catch that really uh could be uh important for catalysis so basically what happens is that you sample all this conformational space most of it is no good for catalysis and then eventually comes a uh a structure that sort of fits into those criteria for reactivity and and and you have a reaction so we've done this in the past for many many enzymes I just put here these this was the very first one in 2012 for HIV1 integrase but then we followed them went on and this is also the alpha amylase as I told you this is just to tell you to show you that it wasn't sort of something that is very strange it doesn't so this follows a pattern so we can say that the small number of barriers accounts for the very most of the observed rate this is experimental averaging so to speak and the average barrier is very similar so these average barriers the the the ones that are good for catalysis are very similar to the x-ray single conformation free energy and that's what we always found if there is not some big problem with the x-ray file obviously uh so going back to alpha amylase we looked into all the structures the 40 structures that we had initially selected um and we saw that these low activation barriers only occurred if a set of geometric conditions was verified and and it wasn't exactly the ones that we anticipated and that we looked for to begin with so what we found was that uh okay and a water molecule that goes and comes back so to speak and this was very surprised because I mean these distances are implicated in the reaction coordinate but this one is not um but so what eventually so we had to study this obviously and we came up with the conclusion that what happens is that this H bond lowers the activation barriers and basically it controls this the pka value of this general acid uh glue 233 and therefore the water molecule switches the enzyme on and off and I say on the time on the nanocyclin timescale because that's the one that we analyzed uh so this is a an example that I wanted to present to you uh to show you uh that these QMM calculations can be very interesting to find all sorts of details uh and just to see what we could find out by averaging over an ensemble of conformations since we only looked at one at a time we went on to do QMM MD calculations on the same amylase with the same um mechanism of reaction and so these are here the QMM results these are the QMM MD results I'll just quickly run you through this so that you understand so here for the QMM these are the results that you get out of that uh MD simulation from which we got the 40 initial structures so the black dots represent the conformations obtained during the MD simulation of the solvated enzyme substrate complex all these black dots and the colored dots represent the conformations that those 40 conformations that we got out of the MD simulation and which conform to those initial that initial initial criteria for reactivity that are pointed out to you and so the the green dots have got a lower activation barrier and the the red dots are those that go up to 30 kilocals per mole and what we found out in running the QMM MD simulations is that if we started with a red dot confirmation that was a bit more out of the the catalytic one still one of those 40 and we had some trouble or not some trouble but it took much more time to make it converge to the catalytic value to the to the the delta g of activation that the real real the one that the best one that we could get and it took quite a lot of the sampling to do that but if we went to a green one for example then we were already there it didn't take any time and to a yellow one it also didn't take much time so we started here and went on and and got the the the the three of the more accurate one so to speak and if we started with the x-ray we are also already there more or less so basically what I want to say is that these calculations are very robust very good and but these others gave gave us enough a lot of detail that we wouldn't have got from this because obviously you can't go on and study many of these confirmations you will get lost in this amazing conformational space that is sampled so obviously you cannot do the two of the time we were actually having a conversation this morning with Peter Freire over this but that really the best thing is to use several methods and see what comes out of this that's how you learn to know your design very very well so we have other the examples that also behave like this so João Coimbra is also doing calculations on protease hiv1 protease which take us to the same the same ideas so to speak and so well I've run used to through all these problems it's now time to to stop as conclusions well to say some of them activation free energy substrate binding free energy and the enzyme efficiency fall in a very narrow range of values for all enzymes as we saw the Hamiltonian has to successfully deal with the nature of all energy contributions if using the ft benchmarking functionals before embarking to mmn calculations is good practice the subset of catalytic competent conformations can be significantly small in comparison with the full conformational landscape of enzyme substrate complexes usually you find x x-rays okay as a starting confirmation if there are no major errors obviously of the x-ray confirmation single confirmation to mmn calculations enable us to understand better the structure activity relationship however many other techniques that explore more efficiently the conformational space of the enzyme during catalysis are used providing a more dynamic picturing to the analysis of the potential energy surfaces associated to catalysis and I just would like to thank first of all professor bill of finance we've got we have my colleague we have been collaborating for years my three this is not all my group but they are the people that contributed the most to some of the things I said here so Rui, Joanne and Nathesia are have got research fellowships they were former phd students of mine and then my two phd students Peter Freire and Peter Piver so Peter did Peter Freire did the AOX mechanism Peter Piver did the MPNO CCST team work and these are not my two students but they are my ex phd students uh so the oracle center martins is now at Scripps and Rita Kalishtu is at the university of Uppsala and well here are some of the people that have been helping us both with money and computational power and well and I would like to thank you for hearing and now you can ask questions if you're interested thank you thank you very much Gloria that was I think really really valuable for people who especially are coming new to this to see this outline very clear outline of you know the different things to take into account the protocol um I'm sorry if it's too long do I have my biochocolic Scherz-Ameriano would you like to ask any questions before I do yeah so while we wait for questions to come in I can already ask a few questions that came up while listening to your to your very nice talk um so when you do the MD simulations to scan for or to look for conformations from where to start the QMM and what you didn't find is you find different barriers however the time to to to make transitions between these conformations is apparently really short in the order of nanoseconds so guessing that the barriers between them are really small so what I then do not so well understand and we also encounter this so it's not that I'm doubting the result it's just that I don't understand why during optimization you then don't cross that small little barrier and then take the lower barrier confirmation that was uh that belongs to a to a different type of confirmation perhaps one is closer to the x-ray structure can you comment I'm sorry you're why can't because I only just heard the very very last part of your question this thing just went off so if you I'll repeat yeah please yeah yeah so I'll repeat the question so in the in the plot of in the in the last part of your talk when you're discussing the the the contribution of sampling conformations there you see that uh if you simulate the the system for a while a couple of nanoseconds and if you're taking different snapshots at let's say nanoseconds apart if I recall correctly you find that the barriers they vary widely my question is what I don't understand and this this is something that that we also observe but what I do not understand is if you do an optimization why does the optimizer not bring you first back to the original confirmation because the barrier must be super super low because it's sampled with a nanosecond so the barrier cannot be very high between these different conformations why does the optimizer not take you first back to the lowest say energy confirmation from where the energy barrier to the transition state is the lowest well because it can be very far apart in the potential energy surface and the uh if you think well they are but it's only 200 picoseconds for example so between 11 and 11.2 there's only 200 picoseconds difference and between well 13.2 and 15.6 only two nanoseconds different you cannot cover so much conformational space in in such a short time I would assume yeah but it with the potential energy surface the algorithm of optimization is not good enough to to do that so when you try to to use a qmnn method that doesn't happen so you you you do the best you can but it's not it just takes a little bit of of change in your activation in your site in your active site to give you a very high barrier uh and uh the the problem is the uh and you can show this mathematically in fact the uh and that's a preconceived sort of a preconceived idea that people do have is that the the the um the catalytic confirmation is the lowest uh that you can find but it it doesn't have to be it can be it doesn't have to be uh in the um in the lowest point so we we it does make a difference and also the the the algorithm that you use and we use several whatever we can uh are not good enough uh to immediately go to the one that you anticipate to be the catalytic confirmation um so uh this is what we get and uh it happens with all the cases that we have looked into uh and uh well that's basically what I can tell you okay the rest now being quest there has been questions I have assigned it to you so I switch off my my microphone now uh Torio says very nice presentation uh regarding multiple PES calculations clustering analysis is going to help in order to reduce the number of structures from a large MD simulation that's a question can PES calculation clustering analysis help in order to reduce the number of structures from a large MD simulation yeah yeah yeah we we we did that as well uh so clusterizing you know and then uh and then um uh truck to get to the uh catalytic structures from there that's something you can do as well but the answer is always the same uh so if you get something that is a little bit different from um from the um um the the catalytic uh distances then the activation barrier is very high um so um that's basically that part of the other part of the question was do you have any study comparing clustering analysis structures with the protocol that you're working with you may have already addressed that um well yes but uh uh yeah uh uh I can if you send me your uh your email uh I can send you some references um I can't yeah that's fine okay we can do that who do you want to put your email into the question then we'll get you in touch yeah yeah that's not a question I think um um the question is can we correlate the calculated free energy barrier with experimental k-cat or k-m values if the rate limiting step is not known if so what is the best way to correlate them and if we know it is a good correlation can we use that as evidence to confirm the identity of the rate limiting step in enzymatic reaction oh yes and yeah well it's it's really the the um rates of reaction they vary so little they they they really are uh so if you don't know them you can always make as I said at the beginning that educated guess because they really most of them are in within the range of 14 kcal per mole and 20 also um that's that's a very very narrow range of values they're all there 80% are following that uh uh in that region so you can correlate anything like you know as soon as you get those values basically hang on I can see Amani's question now so um yeah yeah yeah yeah yeah so uh yes with the iranian law you can correlate sorry actually I understood the another question you can calculate the free energy barrier uh from k-cat so basically what I was saying is that you can calculate the um the delta geovactivation from the k-cat there is an equation that lets us do that iranian's equation so you can also go the other way around if that is your question so if you have the delta geovactivation you can go back to the k-cat as well so another question is in your multiple pers calculations do you impose any kind of restraints in the coordination to many groups of the ts structures uh so uh as I so um I imagine that in your multiple pers calculations you mean by to you mean that in the we are using your we're calculating structure by structure yeah one confirmation confirmation at a time so when you do one confirmation at a time no we did not impose any restrictions in the coordination to the main groups of the ts structures or at least we try not to unless we see that um uh there is or we know that there is a particular interaction that has got to be there uh and then we can restrict it say uh two groups or whatever but on in principle no we don't I can't see anything else okay so I can I can read out uh the question um so uh thanks for the very inspiring talk to somebody else I have a question in case the x-ray structure is not available would you still recommend to perform such calculations using a homology model well um if you have to use a homology model because you really have to um do some sort of project that in which you have to um then you know um it's not such a good idea or it can be difficult because if you make a mistake even if it is a small one with the homology model then you might not get to the correct mechanism or at least if you are in the correct mechanism you might not get to the correct um uh barrier um this doesn't mean that we don't do uh the mechanisms with homology modeling but they always pose more problems than the others that's our experience in a matter so I'm not saying that you shouldn't do it or that we don't do it I'm just saying that it is um we're in for more problems usually um so he responded actually saying thanks it makes sense so thank you for that there's a follow-up question to head of the question uh from Dmitri thanks for the very nice talk right and I have the following question so basically it's kind of following up to the get it question uh so basically when you do first this kind of PS calculation yeah and in the next slides you also show showed the uh what happens if you do QMMMD free energy calculation basically I suppose it's a raw assembly for something like that for metodynamics but for me seems like you can take from at least from the QMMD you can take even a very bad starting structure and after some not so long like vibration time with QMMMD and something you still can get a correct energy barrier no yes of course so yeah if you are you want to see me if I could do the multiple pairs out of the QMMMD simulation my question is what is more expensive and what is should be done in general so you one one thing you can do a lot of PS population well second you can do QMMD long QMMD you you can do QMMMD and still look into single conformations obviously I mean nothing stops you from doing that but the amount that you sample is such that I'm not quite sure how easy that will be so what we like to do is or personally what I like to do is QMMM because I think it's the much more fun QMMMD is more robust I'm not going to dispute that but it's very automatic as well I don't like very automatic things I always find that if you have to think about something you get more out of it but QMMMD or multi meta dynamics or whatever are more robust methods so I think that it is a matter of it all boils down to a matter of personal taste and what you can get out of the methods that you have at your disposition the amount of computational time that you can afford the software that you have available etc etc and that's why you've got so many so many groups with all doing different things to basically get a mechanism of an enzymatic reaction somehow okay yeah yeah thanks yeah so I don't see any other question from oh Gerrit still has a question that you would like to ask Gerrit go ahead you can go ahead well always yeah the question I have is going back to the observation that you find that the barrier is always around but was it 40 kilocalpum all I wonder is this because enzymes have been optimized to work at the diffusion limits so that at a certain point the time at which it takes for the for the reactants for the substrate to actually find the enzyme is becoming the rate limiting step and further optimization doesn't make any any sense from an evolutionary point of view sorry I didn't hear your question okay so this goes back to the observation that all the barriers fall in the same range that 80% or so in the interval of what was it around 20 kilocalorie per mode and I was wondering is that could that could you speculate whether that is maybe due to the fact that the diffusion limit would probably then become the rate limiting step if you optimize the enzyme further so there would be no evolutionary pressure so to speak to actually make the enzyme even faster because then the rate limiting step is the diffusion process of the substrate into the active side so this is what you're talking about right yes okay so this is the activation energy that we get out of experiments so to speak so I think that what it is I mean it's related to the fact that we need the reactions in our body that are fast enough to happen in a very short time I mean if my trypsin that takes hours to calcium amino acids I'm going to have indigestion that's for sure so this is connected the the the the rate of reaction and the the delta G of activation is connected with that you're talking about evolution if I heard correctly yeah so what I meant is that the enzymes have been optimized throughout evolution to reach the point that the rate limiting step is now the diffusion process so that the time it takes for a ligand in this case a peptide or whatever the ligand is for the enzyme to move into the active side so the if the enzyme would be more efficient than that then it don't gain much because the rate limiting step would then be the diffusion would be the diffusion limit yeah yeah I understand that yeah but in fact the it can be also other other other roles can be other physical process can be in play mass flow diffusion mass transfer area all this can come into play now if if you ask me that evolution has optimized the enzymes for the diffusion process I'm not sure if that is the case because I think that with this apparent apparent activation energy that comes to us from hello yeah yes yes we lost you at this activation this apparent activation energy is coming from something so okay so these are the apparent activation energies that come from the experimental values and experience experiments coming from labs that usually do these things now they don't know exactly if reaction rates come from the the diffusion processes or mass transfer area for example that will be into play as well mass flow it can be from all sorts of as well as the chemical processes as well also so it can be several factors and I am not sure and I don't think anybody is if mass diffusion is the major player and it comes from evolution I personally am not very convinced about that I think there are the factors into play that's my idea do you have any idea about that no no but the observation yeah no but the observation I don't think so yeah sorry no no I was just gonna confirm that no I don't have a clear idea either but the observation that barriers are all in the same range that has been observed yeah in several studies so I was thinking there must be something underlying it yeah yeah well it's got to do with the with the fact that with very high barriers you know I mean our our body wouldn't survive but if exactly you know enzymes were optimized for that I think there are many factors into play I don't think it can just be that but you know this is just a speculation good I asked for the speculation so this is fine thank you yeah okay thanks I I have one quick question one last quick question and it's about software so you said that in your group you use both cp2k and orca I was just curious whether there's a preference for one or the other in the context of the availability of particular basis sets particular the functionals or whether it's about what size of number of qm atoms you're dealing with or whether it is to do with your computational resources basically when do you use one which one and when you use the other and why um whenever we use we want to use dp the lpno the ccsbt we have to use orca because it's implemented there and for so that is one reason why we use orca but orca is quite nice to use and we were we started with the cp2k because we started running in oakridge national laboratory and it was difficult to set the other software there and so we were a little bit pushed into cp2k whether we like it or not we had to start using it and then we sort of got used to it basically because you start with one software and you start getting used to it but we run both and probably at the moment we're running more cp2k than anything else but as I said if you want to go into the lpno you have a ccbt you have to go into orca to do that we like them both really provide the answers yeah okay great thank you very much so with that I think we've come to the end of our question and answer session as well so I just wanted to take the opportunity on behalf of you know all of us organizers from bioxcel and all the attendees as well to to thank you very much for the talk I think it's very valuable for people who are coming to this maybe fresh to draw on your experience and you know highlights in different different aspects that you take into account and especially sort of this is benchmarking protocol I think I think it's quite nice for functionals so okay so next Monday we have another webinar coming up that's relevant for this workshop part of this workshop which is agent mahaland will be presenting on the topic of tubers chemical accuracy in qm modeling also enzyme catalysis and also protein vegan binding so it's all lots of good stuff still to come as well next Monday but for now thank you again Maria for for your presentation and for dealing with the go-to-webinar technical issues and yeah thank you very much okay thank you very much okay bye bye bye


still Thursday March 31st and we are continuing with our walkthrough on 287 with Jim. Hi Jim, welcome back. Hello. I actually have a question about I which was the last thing that we I think covered before we are going to go into section 6a. I'm autumn of 13. Oh and did folks catch that sort of deleted one of the versions you might be in the wrong version you need to refresh. Okay. One of them was wrong. No, it was just one of the lengths. So and this was actually Carol's question and she's not here but I'm going to ask it anyway because we're in the section. So the updates to the waiting factors. There's this language here about sort of intentions around recalibration and then there's another section about updates to the weights with a sunset provision. Why are they in separate places? Well, the one I is in statute. So this is an ongoing statement of intent every five years to update the weights and to give a year delay. This was time to adjust to the new weights in their budgets. That's like a prophylactic ongoing statement of intent. We'll come to this yet but 6b the repeal is very specifically saying if you don't do this in the next five years, then the weights will be repealed entirely and it's just a force action within the next five years. So once more we're kind of prophylactic broad ongoing that there's more specific and time bound. Thanks. That's ready for 6a on page 14. Okay. Back to you, Jim. Okay. So again for the record, Jim Damer, that's console. We're walking through S287 as passed by the Senate. We are on page 14 on section 6a. 6a amends the section that you just amended. So this is confusing but this provision goes into effect in fiscal year 29. So this is saying is that we'll come to this later on. There's a transitional period of five years where equalized pupils for any given year is an average of the current year in previous years and it's a five-year average initially, then a four-year average, then a three-year average. That's the transitional period which ends in fiscal 28. And with this is saying, a story in fiscal 29, you're going to stay with the three-year average going forward. So this says that the equalized pupil count should equal the average of the equalized pupil count for the year of calculation current year with the equalized pupil counts that precede two fiscal years. So going forward, it will be a three-year average as opposed to a one-year current. Someone on the side? Yeah. Why are we going with the three-year average and most of the time when we're trying to do producing a two-year average? Why in this case, we're talking about a three-year average? It's like a Passover Seder question. Jim. I'm sorry, what?  Sorry. Jim, do you have a? Oh, that's a policy that was what the Senate wanted to do. So I can't answer as to why they picked two versus three. I think they wanted to smooth out the effect over a longer period, but I'm not sure what the magic is and the number. Jim, I think you said this, we'll see later, this takes effect not for a few years, but is it already, I'm wondering if we're already using an average equalized people are based on ADM at that point still? So we're taking average of an average? Correct. In the sense that in order to get to equalized people, we're using a two-year average ADM which defines launcher membership. So that's how you get to equalized people. What this is saying though is that the equalized people for any given year won't be just the current year calculation of equalized people. You're going to average the current year over the past two years. So in a sense, yes, it's the average of an average. And this is new then.  Correct. Now it's the current year equalized people is used without averaging with past years. Okay. I'm curious to know what the thinking was there, but I will ask somebody else. Caleb? It seems like one effect of this change is it is interesting, Dave, to have something where you've got an underlying two-year ADM and then it's into an EQP and then you basically average a three-year EQP. That is complex. And yet we kind of think back to some of the treatment and this wasn't the same construct, but when we did pre-ADD 46, things like phantom students, that was mostly the kind of slow, the precipitous drop of students. It could also in the case of, say, a district like South Burlington, which tends to be growing, kind of mitigate those changes because, and this is just my take, but I know that our system favors stability over time. It doesn't seem like this change would add a little more numerical stability if there were sort of, yeah, just postulating on my mind. Yeah. Yeah. But it's not just transition. It's ongoing. It's ongoing. And I'll just mention that I had a sort of a similar thought looking at the whole harmless provisions earlier that we've got sort of level upon level of smoothing going on here. Okay. So there is a transition period in the bill, but we haven't gone over it yet. Is that correct? And so before I can understand what the post-transition language means, I at least need to understand what the transition is all about. And that's later in the bill. Correct. Yes. Okay. So thank you. Carol. And just to go follow up with what David was saying, and Kate was saying, is I would like to see some examples of how this numerical stability that's ongoing impacts numbers. Yep. We will ask Brad for that. Thanks, Jim. Okay. Keep going. Okay. So we are on page 14, 6 and 6B, Perspective and Conditional Repeals. So it says, if on or before July 1, 2027, which is five years out, the joint assembly has not revised the weighing factors to reflect changes in cost factors from which the weights are derived. After receiving a recommendation of the new committee, which we'll come on to, to do so, then the weight weighing section is repealed in 6A, which amends that weighing section in the future is repealed. So this is designed to force action to either change the weights upon the recommendation of the new committee, or if you don't do that to repeal this repeal, because otherwise the whole system blows up. Well, trust in our ability to take action. Carol? Well, I would like to flag that because I like that. And then the other thing is, if the whole thing were to disappear, I noticed when we were going through the walkthrough that parts of it would not disappear. Parts of this wouldn't disappear. So would you, we get rid of that, though I'm going to have to worry about all the other places, but if we don't, then there are a number of places that I would have to go back and flag to deal with. All right. Okay. Back to you. Okay. Okay. Now we're on page 15, 6 and 7. And this is the English language learner section. So first we have some definitions. So ELL services means instructional and support personnel and services that are required under the Equal Education Opportunity Act for ELL students and their families, which shall include licensed teachers, paraprofessionals, translate tours, and cultural liaisons, high quality instructional materials such as books and digital resources, family support, and education with assistance from cultural liaisons to speak the student's native language, and community outreach, education, and engagement. ELL students means students who are English language learners, and for whom English is not their primary language. So B goes into the substantive requirements. So B says that each school does each. Jim, before we go to that, where does this definition come from? Came from um, set education or set finance? Did it come from their imagination or did it come from like a, was it, is it like referencing a federal definition or anything like, I don't know why it's about it. I'm just curious if you, yeah. The federal definition is very vague and broad, so it doesn't have these specifics. And this slide was just given to me to use. Thank you. Emily, I'm sorry. Are you talking about one number one under section seven, a sub one, the ELL services definition? No. Yes. Yes. I was asking about the ELL students. All of that. Those are both. And that's true for two as well, Jim. That is, that is a typical definition that I think we use now in statute or as a reference point for this, but with a lot less consequence. Okay.  I wonder if Jim, you could tell us what is the, for how long in your life can you access the technical centers? I think I know what the answer is, but I'm asking. CTE? Yes. Oh, I don't know because it goes into adult education services too, which is less than a wheelhouse. So certainly through grade 12, but I think beyond that as well, under the adult programs. Okay. Then I'll say what I think it is. And I might not. I think Brad has an answer. I'm more confident of answer because he's popped up. That's usually what he wants. Essentially, if you don't have a high school diploma, you can go to CTE anytime at public expense. And whether you're enrolled in a high school or not. So do we need to take that into account in this situation? Because we have a cutoff of 17 years of age in this statute regarding how to wait. I would have to think about that. But off the top, my head, I would say no because I mean, I think I'm not sure where the 17 years of age came from that. Maybe our current statute or maybe federal. I don't know. But I what the students that I was just talking about for CTE who are unenrolled, but don't have a high school diploma, they are their only cost going back to the school district is a partial cost from this from the CTE tuition. They're not counted in their ADM. So therefore they do not roll into equalized pupils. Well, okay, so I'm thinking it might be a policy question because if it's the only cost is their enrollment, that's not the only cost if there are translation services and other things needed for those students, as opposed to somebody who already was just coming in and without the need for translation services or any other things having can they actually read in the language to follow the instructions to be to do what it's needed to do for car repair or whatever else is it happening in the CTE? Yeah, I don't know enough about the CTE programs and such and how they how they put their monies out or how they put their tuition bills out how they pay for things that I don't know enough to answer that specificity. Brad, do you know if when a student hasn't graduated from high school but they're over but they're 18 and over if they then are considered adult ed and they're funded out of it? Where is the funding for doesn't part of that come to the general fund? Yeah, some of that some of that does. I think a lot of I think a lot of them are considered that I think it depends how they choose to go about things. Generally speaking, most students who are out of school and are and are trying to get their diploma in one form or another are working through I why shouldn't say most because I don't know that but a number of them are using I can work through the adult ed program and that has been moved to the general fund. I wonder if you want to do some more research. I know there's something also about IEPs until you're 25 and I don't know the details of that. And another question would be not even connected with CTEs would be how many students are staying with us and for how long they can do that till 22. All right, I'll look up I will look into the entire thing. All right, back to you, Jim. Okay, we are on page 15 at the very bottom of the page subsection B, which has a typo. Just say each school district shall one provide ELL services as defined to budget sufficient resources through a combination of state and federal categorical aid and local education spending to provide those services. Report expenditures on those services annually to the AOE through the financial reporting system as required by the agency and report on educational outcomes of ELL students as required by the agency. Subsection C says that the agency education shall provide guidance and program support to all school districts with ELL students as required under law including professional development resources for ELL instructors and support personnel, information on best practices and wider language development standards and prescribed collect and analyze financial and student outcome data from school districts to ensure that districts are providing high quality ELL services and expanding sufficient resources to provide these services. And then next page 17 changes subject to bit, moving away from required services to categorical aid. So this says that in addition to the wait under the waiting section, a school district that has as determined annually on a double one of each year, one to five ELL students enrolled shall receive state aid of $25,000 for that school year or six to 25 ELL students enrolled shall receive state aid of $50,000 for that school year. E says that the appropriation for for this will be part of the appropriation for statewide education spending. So it won't be a separate line and the payment would be on before November 1st of each year from the education fund. What is the reason for this? Why are we getting we've waited the students at very high weight already something that could be over $25,000 per student. Why are we what's the purpose of this additional and that doesn't even consider federal money. So what's the what's the question? Why is what is this addressing? Jim, do you want me to get that one or do you want to answer? Yeah, it's a policy question. The task force had a number of conversations about concerns that districts with a very small number of ELL students didn't have the capacity to stand up a program that was robust enough to meet the needs of those two to those very small number of students, especially since it's not, you know, say, you know, a district with 10 different schools might have one ELL student in each school that are, you know, 20 miles apart. And the federal money for ELL only goes to districts that have actually a certain critical mass of ELL students. And so this was the way I sort of like covering sort of matching the federal aid money for districts that weren't eligible for the federal aid money. Caleb? Yeah, I mean, and this is a mini grant. So I think we're a really good idea that came out of the task force and waiting the weight of 2.49 is a great it works really well for districts that are at scale works really well in a lot of cases, but you have a really small cohort, there is still there are still some kind of stand up costs that I think can be really helpful by these mini grants. So anyway, I think that both the work of the task force and then some of the testimony that they took in with Senator reinforces I think it's a nice it's a nice add on. The other thing I would add is when we were planning on having a joint hearing with House Ed for this walkthrough and then couldn't find a room for it. But the ELL services and eligibility and all of that stuff is something that they're going to spend more time on than we will. Do I understand that this is a yearly state aid? It's not says to set up your program. Jim, is that right? Yeah, it's an annual payment. And I believe the payments designed to be able to hire a teacher. So it's an annual payment if they meet the requirement. And then Jim, on F, I feel like I've read a bunch of the other state grant statute and this looks like a slightly different language than is usually used. It comes from another portion of Tal 16. It's used in a different place. Okay, so on the subject I was on. Do we have any idea how many districts fall into these one to five ELL students or six to 25 ELL students?  Six to 25 is where we got six students and you're getting somewhere over $25,000 a piece already for those. Not with the 25. A lot of money is. It's not per student though. It's not per student. It's one single payment. The student is the waiting. Yes. We're getting up to a student with the $2.49 waiting. So that's somewhere over toward the $25,000. It doesn't have an amount. Well, you can take the average spending and multiply it by $2.49 and it gives you an idea what the wait will be. And so what the amount will be with the wait. It's going to be a big number. So can we find out how many districts have one to five ELL students typically and how many have six to 25 typically? I believe that Julia has an answer to your question as does Brad. Julia popped up first, so. Sure. Hi. Julia Richter with the Joint Fiscal Office. Brad might have those specific numbers right in front of him. I don't have them pulled up at the moment. I did want to mention that the fiscal note that's posted on the committee page under my name does cite the overall estimated impact that we estimate for only these grants. So in FY24, if the ELL categorical grants were to result in increased spending, JFO estimates this would result in approximately 1.75 million fiscal impact to the education fund, assuming that there's no phase in. And there is a table that has just the information you're looking for, George. Someone just needs to find it inside. I have that table and I can send it to you. I can jump off my screen for a moment and give you some quick numbers. Between 25 and 6, there are, whoops, if I knew how to use a computer, I'd tell you. 25 and 6. There are 18 districts. And between 5 and 1, there are 66 districts. And then there are nine districts that have more than 25. They represent bulk. I can send you something that has that broken out with kind of grass. That's fine. That's my question. Thank you. And Julia is working on a table that has every district and sort of what the poverty percentage is, what the ELL count is. And so we can have sort of a sense of how the weights are going to the data that feeds those weights that we'll look at at some time in the near future. Caleb? Brad, when you said 66 between 1 and 5, you're not counting the districts that have zero students, right? Because that's also a significant number. You're muted. That's correct. Off the top of my head, well, if you give me a sec, I can tell you. But no, I was not counting the zeroes. Before I just throw a number out there, let me just play with the numbers in the background. I'll give it to you shortly. Thank you. You can just send it to us when you're ready. It's okay. Okay. Well, actually, I take it back. What I was talking about here, when I was giving you those numbers, I was not talking to school districts, the data I have are by town, because that's how we were talking about it at the time. That's how I was seeing ELL data. We would have to change it. But the numbers I gave were by town, not by school district. I'll have to... Oh, I know why. Because the data that I had at that point, the ELL data were not by grade. They were just a count. And so in order to go by school district, I'd have to go talk to our data people and see if we can pull it out by grade, which I can do. I'd have done that. But to give you a quick answer of the number of towns, now I got the right word, the number of towns that have nobody, have no ELL students is 165. Well, hasn't it to go back to this, but I guess I'm going to ask it anyway. It's the definition of ELL students. And my first question is that something I've said is going to figure out, or is it something we're going to figure out, or we're both going to work on it? But the question I have is I try to think of a student who is an English language learner for whom English is not their primary language. And sort of who that... I have a grandson who spoke Spanish, but he came from Guatemala, so he spoke Spanish when he started school, but so he was an English language learner, but he certainly had learned it second grade. So how does that... Is there a common understanding of what we mean by English language learner? And is there a moment that you stop being one, even though English is not your primary language? Again, we're out in my field of expertise now, but my understanding... We're always out in my field, it seems. My understanding is that the school districts test their students, and again, I don't know what the proficiently levels are, but at a certain point, they test out of being that, but they're out of being considered ELL, but they stay within, but for other purposes, they're considered ELL for the following two years to make sure that they're okay. So they're still receiving services, and I counted as ELL students, is my understanding. There's an identification criteria that's in some way sort of similar to what an IEP process might look like. I mean, not exactly, but just sort of parallel to that. And then there's three different levels of it, and I think... I sure hope so. Yeah, thank you. But this is broader than many of the other definitions I've seen from around the country, because it doesn't include them sort of three separate levels. Treated differently. Yeah. Kelly. Well, I think the kind of heterogeneity of remarks of English language-language population may play into the breadth of the definition, just because we have so many different kinds of situations with different kinds of... It is interesting this all of our students at school are continuing to learn English, right? You've seen some about literacy and other stuff. It's not, but like you said, there are criteria in terms of the nature of the instruction. Is it considered remedial at that grade level? Things like that. But I do think the breadth, just to my point, is Vermont has a very interesting mix where we do not have just a few kind of groups where you may have non-English speakers without resettlement programs, just some little schools with 10 languages. So we may need some of that breadth to encompass our situation. Okay. Well, that's making me think that back at the categorical aid help, would you want a portion of categorical aid to be attached to each separate language spoken? Because that's a whole other setup. I am hoping, and we'll be figuring that out. Yes. That seems very... I mean, I think part of what's exciting about this conversation on this bill is that we're paying attention as a legislature to ELL in a way we haven't before. And so I think the more committees are thinking about it, the better. That's another thing. But maybe this is really more about ACEs. But if you have a student who's ELL in a situation where they are not a refugee, it's really a refugee thing. Question, which is, I guess after presenting our bill on the floor regarding help for refugees, for the people who are taking in refugees for their property taxes, then I had to read a lot about what is a refugee and so forth. And then asylum seeker and so on. Refugees, by their definition, have experienced trauma. And so I wonder, at least in some way, their family has. So I wonder if that's how to take into account that for providing services or providing help or providing leave? I know Professor Colby will say, and we'll have her back in, will say that one of the reasons that our wait for ELL in Vermont is so high compared to some other states is because of the proportion of refugees that we have in our English language learning population. And I also know that there's a bill that left the Senate, and it's now just got referred to house that a couple of days ago, that's grants for trauma at schools that's fully separate from all of these conversations, but moving on a similar track. Very beginning of that sentence. Yes. What was what did you say that there's something moving through there's a bill that left that came out of Senate health and health and welfare that was put it out of the Senate and got referred to house at that is for trauma instruction or trauma support grants in schools. And I hope we've finished our walkthrough by noon, so I'm going to go back to Jim. Yeah, sorry, Scott. I'm on the top of page 15 section 74013 a sub one sub ABC and and I'm trying to look at this US code 1703 but a B and V especially that if I was a business manager, I would look at that I would say all of my licensed teachers and professionals and all my materials that they're being used for ELL and ELL student that I could I could assign that to that ELL category. But the reality is is that ELL student, many of them, they will be in general education classes with a teacher that is not an ELL licensed teacher, they'll of course circle back to a class where they do have an ELL licensed teacher, but that language right there looks to me to be so broad that a district could almost assign any expense to an ELL category. So I'm just kind of looking at that. It looks really broad. I'm just wondering though, the context of what we've got in front of us whether whether it matters whether the school is going to get the grant and they're going to have the weights regardless of how the code that costs. Well, ELL is a protected category. And I think their requirements are a little bit steeper than just giving them a later, giving them money. And that we actually keep it, say Vermont, we actually keep track of how much money districts are spending on ELL service. It's about the only thing you keep track of to be honest with you. But in terms of what we're looking at here, I agree with you, but in terms of what we're looking at here, would it matter? I think it does because I think it creates a loophole where a district could just assign all general education expenses to ELL, which may short change the kid on ELL services. Let's flag that for now because we're going to have more time this afternoon to have a discussion. And so I want to, if we can keep discussing that. Okay, Joe, and you're muted. Sorry about that. We are on page 17, section eight at the bottom of the page. And this is taking out small school grants entirely. I'm leaving in merger support grants. So if we go to page 22, so page 22, it's just striking out the small school grant statue. And now on page 22 is replacing that language with what's here, which says an A that a school district that was voluntarily formed under F-46 and received a merger support grant. So continue to receive that merger support grant, subject to subsection C, which I get to. B says if you were involuntarily merged, you get your merger support grant as well. So whether you're voluntarily or involuntarily merged, you get the merger support grant, except you don't get that if you get a weight. So if you get a small school weight, you don't also get the grant for that year. Maybe it's a Brad question. Do we have counts? Do you have counts at your fingertips, Brad? How many districts would fall into these categories? Not at my fingertips. I certainly can get it, but I don't have a handy. And that table that Julia is working on will include exactly which districts would get the small school weight. But we can see who's getting the merger support. These are at the district level, not the school level. The small, sorry. The small school. Wait. Is that at the district level or the school level? At the school level. The sparsity is at the district level. The small school is at the school level. And that's why we do that funny enrolment counting thing that we talked about. The grant, however, which I'm not sure if this is your question. The grant goes to the school district. And just even though they're counted at the school level, so you would only get that weight if you had a small school. Of course, the EQP are ultimately counted as district level. So those small school, those kids in a small school, ultimately, their larger weights, which only they get, will be aggregated into an equalized pupil number that will apply for the whole district. So it's kind of a mix because ultimately the rubber hits the road in the district budget calculation, but that additional small school weight is only accrued under that certain schools according to the categories. But ultimately, it's all the EQPs locked together and handed to the district during their budget negotiations. Jim or Brad, do you have anything to? No, I think what Representative Elmer said that is correct is that the weight itself is calculated on the two-year enrollment, two-year average enrollment of the small schools. But then that weight gets brought up to the school district level and goes into the equalized pupil count. Thank you, Jim. Okay, we are on the top of page 23, still with this Mergers Support Grant. And all this does in Subdivision 2 is the Mergers Support Grant goes away if a small school upon which it was based is closed. Okay, so section 9 and 10 are just conforming changes to get real references to the small school grant. So that takes us to transition at the bottom of page 24. Section 11. So this provision reads that for fiscal years 24, 25, and 26, so the first three years of this transition to the new weights, the number of equalized pupils in a school district shall be determined by averaging the equalized pupil count for the year calculation with the equalized pupil counts for the preceding four fiscal years. So for the first three years of transition, you have a five-year average equalized pupil count. For the fourth year of this transition, fiscal 27, it moves to a four-year average. And then for the last year of transition, fiscal 28, it moves to a three-year average. And then as we talked about earlier, for fiscal 29 and thereafter, it stays with a three-year average. And Brad has a table of this that we can look at after the walkthrough because I don't know about you, but it's pretty hard to visualize that. Okay, section 12 is another transitional section, providing that for the transitional years, those five years, the excess spending penalty is suspended. And for those five years, the 3.5% whole Thomas provision is suspended. We skipped two sections of current law, I think. And section one. Jim, just two sections. I'm just reading the current law, and I have a five-plus two. Fascinating. Well, it affects my district. Back to you. Oh, okay. Okay, section 12A is the next transitional provision. The ballot language for budgets has specified language and statute, and this suspends that requirement for the five-year transitional period. Section 13, page 26, just requires that the Vermont Center for Geographic Information work with the agency to determine the number of persons per square mile in order to figure out that weight. Jim, I'm a little bit behind you maybe explain this, but section 12A suspends the ballot language requirement. What's the thinking behind that? I have to speak on behalf of another member. To answer that, my understanding is that the way that's done now on the ballot, there'll be a lot less control of school districts in terms of where the tax rates are going and the reasons why they're changing during those years. And to use the current language that's required in the ballot, I believe Senate Finance, that would be confusing to people. I believe this would leave up to the local school districts to determine what they put on their ballot. I have that thought personally, I think that maybe can use some, yeah, a lot of the, yes. So listen to the four report, Senate four report, and basically the report was that because there would be swings in per people spending that were that were part of this and not that were because of this bill, that they didn't think it would be fair for the districts to have to report those percentage changes basically. And then Jim on section 13, do you know if the Senate had the GIS folks in? I don't recall. You're back to you. Yeah. Okay. Okay, so page 26, section 14. Are we through this? This is the requirement. So it says on before December 15, 2029, the state auditor shall submit to the House of Senate, Community Education, Ways and Means, Finance, AOE, and the New Text Advisory Committee, a performance audit conducted under generally accepted government on other standards that identifies the successes and failures of the implementation of this act, including whether and to what extent each of the acts five goals under section two have been met. If a goal has not been met, the reasons why and recommendations to achieve that goal and the fiscal impact of the act, including the cost of implementation requires that before December 15, 2024, the auditor, the AOE and the new committee will jointly agree to the statement of work for the audit, including how to measure whether the acts five goals have been met and submit the statement of work to you. And then B on page 27 says that the audit shall be carried out by the state auditor or a contracted designee who in order to maintain independence has not consulted on or contracted to provide services in relation to the people weighing fastest report or the report prepared by the task force. The art will cover the period beginning on July 1, 2024, and ending on June 30, 2028. So that's the implementation period. The art shall take into account such metrics as the auditor, the agency, and the committee jointly determine appropriate, which may include one school district progress and the EQS standards. I'm sorry, before we jump into that, I noticed that JFO was not included in any of these lists.  I don't know. This was the language I was asked to draft. So thank you. Okay, we're going through the list that the metrics that may be included. Number one, school district progress on meeting EQS standards and other relevant education standards, such as the wider source of standards for English language education, Common Core, and next generation science standards. Number two, student performance progress on proficiency-based learning assessments, graduation requirements. Student performance progress on standardized tests, such as SPAC, new and common assessment program, et cetera. Number four, Vermont Youth Risk Behavior Survey results. Number five, graduation and post-secondary education enrollment rates. Number six, education spending and homestead tax rates. Number seven, education for compensation levels and full access for staff and eight academic extracurricular and student support services, resources across school districts. It requires that the auditor host the webpage that provides transparency to the public on its work for the period July 124 through July 1629, which will include when available the following informational links to this act, the statement of work, reports made to you and other public bodies on its work, and all metrics used in the audit. I'm curious to talk to the auditor about their office's interest in this project and sort of the parameters of it, but I also imagine that the education committee, given that most of these are education metrics and not finance metrics, will have a or educated eye on this. Any questions in the section? Yeah, I wanted to flag that I have concerns about it. I have concerns about it, sorry. And so I want to flag that. Just general. Yeah. Yeah. Okay. Back to you, Jim. Okay. All right. We're on page 29. And this is section 15, it's creation of the Education Fund Advisory Committee. Membership, seven members, Commissioner of Taxes, Secretary of Education, or Destiny's, and then five members of the public, all of whom have expertise in education financing, two appointed by the speaker, two by the committee on committees, and one by the governor. Powers and duties are amily on report January 15. The Commission might recommend recommendations to you regarding updating the weighing factors, which may include recalibration, recalculation, adding or limiting weights, or a combination of these actions as necessary. Changes to or the addition of new or the mention of existing categorical aid as necessary. Changes to income levels eligible for property tax credit means to adjust the revenue sources for the education fund, including whether to transition to an education income tax means to improve equity, transparency, and efficiency in education funding statewide, whether and when to reinstate the excess spending threshold, and if reinstate, if reinstated at what level, whether and when to reinstate the ballot language, and if reinstated at what language to use, and the amount of the stabilization reserve. That says the committee shall recommend updated weights and categorical aid at least every five years, which may include a recommendation not to make changes where appropriate. And it's first report to you next year. The committee after consulting with the Department of Taxes, AOE, and JFO will make recommendations on the implementation of the education income tax system to replace the homestead education property tax system, including implementing a retro tax, retro tax credit, or other mechanisms to ensure from others who pay rent, who rent a primary residence, participate fairly in the education income tax system, means for a minister during the new education income tax system, and ways to transition from the current homestead education property tax system to the new income tax system. Assistance is from the Department of Taxes, AOE, JFO, this console, and Office of Ledger Operations, meeting to occur first meeting by July 15 of this year, and compensation is usual per DM and reimbursement. 15A is the appropriation for that, which is $2,500 for fiscal year 23. Section 16 is a session law requiring collaboration by the agency. Before we go to 16, are these lifetime appointments? I didn't see a term. There is no term here. Section 16 is collaboration between AOE and JFO. It requires the honor before August 1 of this year. They enter into a memorandum of understanding to share data, models, and other information that's needed to update the winning factors. They have to host the model used to provide modeling for the winning report, and to ensure that's updated and held in parallel, and they need to recommend, based on their consensus view, updates to the winning factors to the Education Fund Advisory Committee on a scheduled imperial basis to accomplish cost changes underlying those factors. Then section 18, these are now conforming and technical changes. Section 18 takes out a few categories of exceptions to the education spending penalty that are not being used. The first one is exception for spending triples of the cost of playing a merger of a school with fewer than 20 students. Second is cost associated with dual enrollment and early college programs, which are not paid by the school district, they're paid by the state. Section 19 is just a small conforming change, equalized pupils, clarifying that it's for the year in which it's calculated, because now it's being averaged over various years, so this just clarifies that the base term is the year for which it's calculated. Section 20 on page 34 is just referencing, let's see, this is in the power of a school board, former vote, it's just referencing how equalized pupils are determined under the section we went through earlier, so it's just a cross-reference. Then effective dates, a number of these take effect on passage, but the parts that take effect until I won next year, which is on page 36, sub B, are the parts that deal with the weighting changes going forward, so there's a one-year lag before those changes take effect. And that is it. Thank you. Apologies for losing my voice there for a while. I realized a moment ago that you, while we were taking a break, you were jumping to another committee, so I'm really sorry, Jim, this has been a bit of a long haul of a warning for you. It's okay. So here we are. So we're going to have a longer committee discussion about this afternoon. I think now it might be helpful, and of course I think we've already flagged the effective dates as something that might need some mapping out on our, so I think switching over to Julia to just take us through the fiscal note might be helpful, and then maybe if we've, if we still have a minute looking at Brad's transition table. Julia. Good morning, Julia Richter with the Joint Fiscal Office. The fiscal note for this bill should be posted under my name on the committee page. Do you want a moment to pull it up or should I start walking through? Give another five seconds here. See them coming up. Okay. To you. Thank you. All right. So the fiscal impact for fiscal year 23, JFO estimates this bill would result in approximately 2,500 to the general fund due to that appropriation for the Education Fund advisory committee. With respect to the Education Fund, it would have a fiscal impact in future years. So as I referenced earlier in FY24, if all ELL categorical grants were to result in increased spending, we estimate this bill would result in an approximately, approximate 1.75 million fiscal impact to the Education Fund. And this, this stems from those categorical, that categorical aid with regards to ELL. And as the committee is well aware absent any other changes in policy, this would result in the base Homestead yield and or the base non-Homestead tax rate to need to be adjusted to account for this anticipated increase in categorical aid. The rest of the fiscal note, the next couple sections just outline how we at JFO generally estimate per DM costs, as well as currently drafted, there are no anticipated transition costs to the Education Fund or general fund. But I really do want to flag this last section of other potential fiscal issue. Sorry, Julia, before you get to that, there are no audit costs in there because there was no appropriation for whatever contracts the auditor would need to issue. Yes, that is correct. And also because the, sorry? Do you know if the auditor weighed in on that? I believe that Senate Finance did take some testimony from the auditor. I am not sure what, what they said as the bill is currently drafted. I believe the audit would occur in fiscal year 2029. I don't know any more details about what costs are anticipated in the future for that. Are there any other questions before moving on to the potential fiscal issues? Okay, so, so this last section is just bringing up some issues that I think that the committee is well aware of or the adjustment of people weights. This bill will shift tax capacity for most school districts and towns, meaning that with constant education spending, some towns tax rates would increase while other towns tax rates would decrease. In other words, assuming all education spending remains constant, towns that would have fewer equalized pupils with the new weights than they had with the current law weights would have higher tax rates, the converse also being true. This will likely have an effect on the education fund, including the property and income yields, but because total education spending is determined by local votes, we can't, we don't know what will happen. It's potential, it's total education spending may increase, but it really will end up being determined at the local level. So that's why the impact to the education fund remains unclear at this point. The transition costs, we've got obviously the one sentence saying that there's no anticipated transition costs still in this context would mean by transition costs, just to call it as I understand it would be four districts for whom this change would result in a higher tax rate under the change. Transition costs in theory would help to mitigate the pace of that tax rate change. So in other words, for districts who would have their tax capacity reduced and would normally have a higher rate, we talk about transition costs, if they were to put them in, they would basically go to defray or slow that rate of increase. Is that right? I think that that's really a policy decision, that it would be up to the committee to determine the best transition mechanism and figure out if, depending on that transition mechanism, figure out where those costs would fall and who would be affected. And just to follow up, given that it's a policy decision that hasn't been made, how come you've got this section in here? I guess because there's nothing, we're not doing anything and it sounds like it's a little undefined that this is a policy. I guess what's the utility of having this sentence on transition costs at this point? Well, it was decided by the Senate, right? So the fiscal note matches as the bill was voted out of the Senate and so... Did the Senate make a transition cost policy? The Senate made a decision not to have a transition cost as they've, as the bill reiterated now. So it's kind of calling out the absence of a policy, is that right? Is that what this is? The transition is just average and weighty, so there's no dollar assigned to it. So there were some, if I may interject, there were some questions in terms of interest in us clarifying what the transition cost, if any, overall, what that would be. And as the bill is currently written, there has been the decision to use the transition mechanism that was earlier presented by Jim in terms of these averages moving over time. With the different years. So for that reason, there hasn't been source of an additional cost that would be used for that transition. It's up to the committee and ultimately the General Assembly to decide if you want to change that transition mechanism. But as currently written, yes, we do not anticipate there to be any additional transition costs. And so this is referring to the three year equalized pupil weighting. It's saying that transition mechanism of having a three year average does not cost the state money. Is that an accurate way to look at this? It's referring to the transition from current law into the proposed new weights and saying that as it is currently, it's solely shifting the tax capacity within school districts. So there's not, as the bill is currently written, there's not any other provision that would provide additional money or change the cost as it transitions in. It would just be transitioning within the school districts. Julia, looking at the backup to the section on the ELL grants. And you qualified this by saying if. But it also seems possible to me that that money is already being spent. We don't know. But the districts are already including that in their budgets so that it's possible that it wouldn't result in either as much or even any increased spending. Yes. So the reason there is that qualifier of if it were to result in fully increased spending, that would be the cost. That's one of the nuances of the education fund that I know you and the committee are well aware that because of the nature in terms of funding of schools and funding of these different programs, we don't necessarily know what the local decisions will be. So this, so as you as you rightfully stated that that that estimate captures if if it's all increased spending. Thank you. Thank you very much, Julia. Yeah, I had another question. It's back to Jim. I don't know if we want to leave this, but can you save the question? Actually, you know what we're not going to have Jim for the first hour of the afternoon. So yes, ask that question to Jim. Jim, if you are there. Great. Jim, my question is about the annual report or deliverable from the new Education Fund Advisory Committee, January 15th. And whether that would replace or supplement the December 1st letter. I'm not sure if it mentions the yield in here. Maybe that would answer my question. But is it going to be in lieu of that letter? Or in addition to? In addition to, yep. So it's not it's not intended to be used for school districts to set their to help them with their budgeting, necessarily. That report is really to be used is for you as your assembly in order to to make possible changes to the rates, etc. So it's not really about yield. Yeah. Okay, good. Thank you. I'm thinking that trying to look at this table of the transition in 10 minutes or less is not a good idea. I'm listening from that laughter that folks agree with me on that one as Brad. Great. Okay. So we will thank everyone for getting through this together and see folks back here at 115 for to talk through this table that Brad has posted and to have a general discussion of the morning.