 In druga aspeta, ki obstavljeni skupni semulacijo, je to zvukovana in zeločenja. Zato, kako počerite o nezavršenju, ki ima vršanje zvojo Bela Léna-Jone in eletrostatike, to zeločenje zeločenje izpravila in izgleda, ki v svojo zeljev, ki je obstavljenja, in zelo vse odličenje, ki je odličen, kaj je odličen, z dve, od tri, odličenje, je vse odličen. Zelo da se imamo, da, kaj smo v sestu, vsega in nr. odličenje, smo vsega in zelo vsega in zelo vsega in zelo vsega. Vsega in zelo vsega je vsega in zelo vsega, da smo vsega vsega in zelo vsega. je zelo 90% kompetencija. Zelo smo zelo, da nekaj potenšel, nekaj Lenardžan potenšel in Kulom potenšel zelo več več več več zelo. Zelo, da je zelo, da je tukaj potenšel tako, da smo zelo kompetencija. Kaj je potenšča, kar je zelo zelo, je zapomeni, da je zelo zelo, da je nekaj kutov, nekiče zelo, nekaj nekaj zelo, nekaj nekaj zelo. Taj kutov, ko je vzelo, če so počkaj vzelo, wojte je zelo zelo zelo. Be le feste se uizovati vse, za to, da se veči izgleda, da je ovo zelo zelo izgleda za kaj je zelo zelo. Kaj je vse početve početve? Početve, kaj je to zelo, ne bo, da se početve vse vse mežimo, ne bo, da se početve početve, in vsega je zelo vsega zelo, ki se načeče načeče. Načeče je to načeče, ki se načeče vsega je zelo, kaj je zelo vsega zelo, ki se načeče, in za toga opščenja je začeti vsega, da se je vsega vsega vsega vsega in vsega vsega vsega je parametračnja. Tako vsega parametračnja ne bi nekaj arbitari. tudi jaz sem zapravila. Kako jaz sem zelo, neseljeti pa dobro vrst, ne poči očite, ne poči očite tudi zelo, bojne zelo. V zelo, kako nosimamo in vse vse sprem, vse sprem nabakite, zelo se na vokadno tako, da imamo 10,23 molekul, tako, nekaj molekul in kontakt s bordem. Kaj smo simulati molekul, in pošli smo to v box. Tukaj molekul automativno, tukaj, da molekul imedaj vse vse vse vse vse vse vse vse vse. Tako, vse vse vse vse vse vse vse vse vse vse vse. »Don't water gum rats in controversial water This will affect our results, and impedim envah Rickham, was passing by wild of the ball than this way we will not want because then we hacerlo will affect our results that will be an environment very different from the molekul inside and bigger in the experiment. a so we use boundary condition, in peculiarity our box is, in the picture is the central one in red hodnji država, kater je nekaj kot pa i nekaj pa iz vsej oven, na vsej soli. To je nekaj zelo, je našliek. Poje listsu igreš im me nekajbe za volje ovoje in in da je tega pravda vzela, nače ne želimo. To nekaj tega za to, če zelo. Kako zelo odvijevamo na kot, najbolj je, da je zelo odvijevamo na kot, najbolj je, da je zelo odvijevamo na kot, ali bodo, da je odvijevamo na kot, da je nekaj nekaj delovodnji kot. In je zelo zelo od nekaj komorov, nekaj komorov, nekaj makromolegular, nekaj komorov. Zelo prizve, da je zelo bolj, dveče je začal v žezrstv. So what is the problem if we use a cubic box, we might have too much solvent to account, so too much interaction to calculate that will slow down our calculation without providing extra information. So we have in Gromarx been developed different type of box that can, that I helped to reduce the volume of the box compared to the cubic one, so we have the X-analon one that can What emission do we have used for the membrane simulation or for more global protein we... one can used to create row glitter charitable or top Ronytic to echo magnetic, for the example boss we have a reduction of 87% of the cubic volume while for the other 2 we have a reduction of 77 or 51 And when you built the box you always have to take care that the distance And is following im, is large enough that doesn't see each other. Otherwise you have an effect, if your proteins seen your protein, it means that it might effect and bias your results. It's important that the distance between the proteins at the edge of the box should be at least large and then twice the cut off. MORE Sorry larger, so it's important that the distance between your global prote疑 and edge of the box is larger than half the cutoff. IMPROVANT TIME STEP The integration time step determines how much time can be simulated. If you have a small time step you simulate tudi je sljedačja oceljava. Kaj pa neččetno je tudi mstavlja, jaz lahko z njega v pošličenje tudi konflotationalna tita, tudi mstavlja, tudi ljubna ljubna ljubna. Tudi mstavlja je nečetno je vzil, ki jaz poljim tudi ljubna ljubna ljubna ljubna ljubna ljubna ljubna. In zelo lahko izgleda nečetno, nečetno je nečetno izgleda. Vse čeknima ljubna ljubna ljubna ljubna ljubna ljubna. the time step you will get instability so you will get problems in your results. So we have to have an appropriate time step so if we consider flexible so if we consider vibration the bond vibration and the rotation the torsion and translation all this component type of motion we probably need to use a time step of one femtoseconds if if we you want to z vsem, da je tukaj toga, rotacija in vsega, potem vsega na 2 femtoseknos, in, da je tukaj vsega na femtoseknos, potem vsega na 5 femtoseknos. Tukaj je vsega vsega vsega in vsega na potenčno. Kaj je nekaj potenč, nekaj nekaj potenč, nekaj nekaj nekaj potenč, nekaj nekaj nekaj nekaj potenč, nekaj nekaj potenč, nekaj nekaj potenč, nekaj postoč nekaj potenč, nekaj nekaj potenč, nekaj nekaj potenč, nekaj nekaj potenč, vsega vsega na nrten, na to, da je zemljene lak dynastyk. Tkaj nekaj lak nekaj lepo dan aviatom. In nekaj nekaj zelo, da je izgleda algoritm, da je izgleda tvoj bolj. V gramače imamo links, p-links in však, da je zelo. Zelo, da je zelo, da je izgleda tvoj bolj, da je zelo, da je zelo, da je parametrizat. Nelj, však, da je vse vse hodin in metilim grubem. In zelo, da je opšta, da je to vse, da je zelo, da je vse, da je vse, da je vse, da je vse, da je vse, da je vse, da je ciligravče zelo. Samo Bugjeva imamo izdemih, da je zelo, da je tvoj bolj, da je vse, da je vse, da je vse, da je vse, da je vse, da je vse, da je latem. in dokjem je zelo srečen v prišličenem zelo. To je moj najzavšoj zelo, da je bilo tudi v fizika in kemikalji, v biofizika in biofizika v biojusih, je vzelo v mpt in zambal, zelo vršanih zelo vršanih, vršanih zelo in vršanih. na koncu glasbenih, njomovih, poznjovih, poznjovih, poznjovih, poznjovih, poznjovih. Tudi, ni sest, da vročim, da ne piszem tudi prav fragtučnimi ili začala močim. z pristimulacijem, z pristimulacijo. Kako se počeš? Zelo smo počeši drugi počeš, tako, da je prišlo, ne odličimo v vseh, neč zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, imaš vse zelo este. Suprem pridlje in simulacije raises z kanetikon. Kanetika je od njega pridljeva na otvarno vsega sebega. In oddona je značno entre kanetika in nr master deepest in Danes. In ili se vsega Hitam konectične energije, kaj smo pošličili temperatičnje. Znamenja, da KB je boljstvana konstant. Tukaj, kaj je tukaj, kaj je izgleda, izgleda temperatičnje in simulacije, je z modifičenju, očeljenju, velocitivnih vsega. Zato, kaj je izgleda vsega velocitiva, Getting to well, how to be changed, where we can affect the temperature. Pressure and simulation are calculated in a different way. So from the ideal gas theory, we know that there is a relation between the pressure of the system, the volume of the gas, the number of particles and the temperature. For a real system because we are not simulating ideal gas. For a real system to this equation we add a second term. in taj term je derač, vzlizovacja se s vrčenjem dvečja, in poziracja. Zdaj, da je poziracja vzlizovacja sezvorjeva, oprešljenja, preloženje taj držav. V svoj tempore, in ki prizvam vzlučovacja v molekularu, je, da bi vzlučilo poziracja kaj je molekul, in v ovom različenju skaljamo voljum. Zelo možemo, da bomo pošličiti kristal cel. Zelo, da bomo pošličiti, da bomo pošličiti kristal cel, in zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, da je zelo, that the molecules as a in the in vitre experiment or sometimes also in the cellular environment. That means we have to add to our macromolecule to solvent, ion with correct concentration. If it is a memem protein we may add also a Membraine mimic as a bulayer, Lipid bilayer and other atoms and other molecules that might be an important part of the environment. And when we choose those dissolvent models, the ion models, the lipid models and whatever other molecules models, we have always to do it in line with the model, that we use to describe the macromolecules, so in line with the first field used for the macromolecules. That means that you should be all compatible. And that is will be the all box that you will see, simulated box. But we still need, for the microorganin particular, we need a starting configuration, but also maybe for the solvent we also need a starting configuration. So for the macromolicle usually the starting configuration comes from experimental data from structural biology, from x-ray crystalloffs, INMR, spetroskofi, krioletron mikroskopi. Z njega je tudi izgleda tudi tridimensjonalne modele, tudi na modeli, modeli in dokim modeli. V zelo solventu, vživljeni, smo pre-gleda solventi box. Zelo bolj bolj solvent, je pre-equilibrati, in je zelo zelo, zelo vzelo vzelo vzelo vzelo v mikromole. Lakšno, že v makromoleko, kaj si naredila pri PDB-data bank, lagš robbed, kaj si izgledo nekaj preddaj beach, več, ki se priče zelo na zelo, na hrv. In idem, da si počutila na to, počast bo, ki za vse potreboj, quando se nada kalec naredila pri PDB-data base, je zvaril inse. Perquè potem je če mojno izgleda, ne billionje ne mora ste zelo, je ampak izgleda, da se je vse zelo izgleda, neč je obsudne, da bi se počnosti, ko je tako začno je čest, tako na različno. Zato, ker je tudi kajrat, da ne všim zelo vše applizacijanom vse vsega obsudnja. Se držav, vsak, svekben sva. Kako pa ne vsak v tečniši uniti ne viče, struktur, kar je vseč ne. In vseč je vseč, ko je čestno vseč, in ne bo rov data na toga, ker vseč ne vseč, vseč vseč je vseč vseč vseč z rov data. Vseč je poslutnje idroga. Vseč je poslutnje idroga. Vsič je poslutnje idroga. Vsič je poslutnja idroga. Vsič je poslutnja idroga, kaj smo videli, na kaj je poslutnje struktur, na modelu, od zelo refinovih modeljih. Tako, bo zelo, ki se da bo, da priživamo poslutosti te vse atom. Tako, da bomo razredno modeli na nekaj, ki smo odrani na strašnji. Da so prizvečili, ki mali modeli te atom, sem tudi bomo se vse pahne, ko je ta strašnje. Zato je vse pahne, kjer je te proprzito, korrekt for each residue, if you are a protein, for example. But it could be that in some particular, for example, in a pocket, in a protein pocket, you might have an effect of PK shift. So a side change that usually is the protonate and that pH might be protonate or other way around. So also this has to be think about. And also one main thing is to account for possible tautomatic states. Tautomatic states are very difficult to detect experimentally. But for the protein, we have only one case that is the histidine that can have two tautomatic states. But in a structure like RNA, for example, it might be occurring more frequently. So some thought has to be spent there when you set your simulation. The position, the water position. So maybe in the structure there are some kinetic rubber water. Maybe those things that are built as a water, if the structure in particular is a little old, might be not water, might be ion. Also you have to pay attention that sometimes extra molecule are present in the PDB and those molecule are there just to allow the resolution and the detection of the structure. So that I mean cofactor, lig and sulfatane, for example. Or also you have to pay attention to special conditions that have promote the servicional splinter structure like pressure, temperature. So that are all aspects that you have to think about before setting your system and just using the structure that you get for the PDB. So one other useful tips is to read carefully the article from which the structure come from. So we have now the initial coordinate but we would like also to have initial velocity. We might want to have. So if all the velocity at time zero at zero means that your system start at temperature zero since we know that is the relation between the kinetic energy and the temperature. So one way to generate in itself velocity is to generate the velocity and distribute them in a random way on all the atoms. Such a way that we start with the system at the correct temperature. Might be the distribution is not correct but the temperature is correct. In other case, if you have a previous run you can take all this information from the previous run. Molecular simulation. So we say that we need we have a starting structure usually coming from an experiment or whatever from a PDB structure most of the time. We have to choose to assign a functional form. One also good tips to know for this define a functional form is to look how much people are using that special functional form. More people are using a force field. It means that more people can directly check the force field. So you can trust more the force field for that specific property. More people use the force field in particular for that specific property. Then we have to add the simulation condition that means temperature, pressure then also to put our molecule in the correct environment that we want to simulate it. And when everything is ready we can start molecular dynamic simulation and generate conformation. This ensemble of conformation that at the end we get an ensemble of structure from that ensemble of structure we can extract thermodynamic property, structure property, kinetic property and dynamic property. Now I want to give you an overview of the file, the extension of the file that are using Gromax. So structural file are mainly labeled as Grom file while file containing information of the potential of the energy function, the potential form are called topology file, the extension is top and file containing the parameter for the simulation are mdp file. And these three files are the input, the basis input for the simulation protocol. Then when you run a simulation after you run a simulation you get an output and this output is characterized mainly in the main portant file that file collecting all the trajectory that is called xtc file, the trajectory file contain all the structure that you have generated and are saved according to the frequency that you have set in the mdp parameter because in the simulation parameter you set also the frequency for which you want to save your trajectory, your energy why we need this because we cannot save every step since nowadays we will run in the order of microseconds and we cannot save every two femtosecond file become too large. So we have also an energy file where we store all the energy, a dr file and we have also log file this and then we also get a last step as a grow file last snapshot. So this is input output in the groma thought with the correct extension, more details on this you will see in the tutorial after the lecture. So the first things that we have to set for simulation we have to set the system. So the system we have to choose the initial coordinate maybe we visualize the structure we add missing atom and we have our first structure then after that we have to generate a topology all the atoms of the macromolecules then we will define the box of our system and then we add the environment so first we add the solvent and then we will add the ions. So of course when you add the solvent, when you add the ion, all the model for the solvent ions should be consistent with the model for the macromolecules and in this phase probably you have also if you miss some parameter you have to generate a missing parameter. At the end when all these steps are done in this order we get the starting configuration that we can use for the simulation. Before the data production we need to relax our system in different steps this is the step that we suggest to do it so first of all we will minimize the energy of the system that means that so to to remove some steric contact to start to relax the system then we will run we suggest to run position restrain calculation to relax the so we position restrain the macromolecules but with us the position of the solvent and the ion that is important since the solvent is just coming from a prebuilt solvent box so it doesn't when the molecule is solvate the orientation of the solvent is not the most favorable orientation respect to the macromolecule and the ion are just other replacing water molecules so we might have ion too close to the macromolecule that should be never be there so then it's important to relax the position of those items otherwise we have a deformation macromolecule from the beginning and that is very critical in particular in system highly charged system where a charged player large role or in system in nuclear acid might be more critical like RNA and DNA the other things that we have to get is our system to get the system to temperature to the correct temperature and pressure condition when all this is done we can go to the last step of simulation and this simulation so where we release everything and we start really the data production from which we get ensemble of conformation from which we can calculate the kinetic property each of these steps are performed with a dedicated mdp file then we come the things when you finish the simulation you can check that your energy is okay that the distance between the molecule is large enough but then I suggest you to visualize your trajectory because with the eye you can understand better your molecule and you can also realize which type of property and which type of atom might be which kind of interaction might be more interesting to look in it or not but it is just the person suggestion and there are different tools for visualization and as we must use vmd and that you will see also in the tutorial after that I thank you all of you for the attention and after this talk we will be question and session for question and after that we will have the tutorial. Thank you, bye