Hello everyone, I once again welcome you all to the last lecture of MSP lecture series on Transmittal Chemistry. This lecture I shall consolidate whatever I discussed in last 59 lectures in the form of summary and conclusions. To begin with what I did was I gave a historical background to the periodic table why I gave it is very important to know how many of these people contributed to bring all the known elements in the form of a table to understand their chemistry and also to do comparison and also giving some order as I had mentioned periodic table is nothing but place for every element and every element in its place. So meticulously it was done and it was published by Dmitry Mandeleev in 1869 but however at least 50 years before this concept came into the mind of Dmitry Mandeleev several other all chemists or physicists chemists in general scientists worked very hard to arrange those elements in a organized manner in that context. I call all these people as periodic table scientists you can see here among them who stands taller if you ask me it is Antoni Levasier he changes the perception of understanding of science and chemistry at the time when people were not going in a different direction to propagate the advancement of science at that time he brought the concept of quantitative analysis instead of qualitative analysis and he can be called as father of modern chemistry and of course he named oxygen and hydrogen and also he named several other elements he also discovered silicon and unfortunately at the age of 51 he was guillotined. Next Mandeleev published his periodic table in 1869 but before that triad system came into picture by John Newland and then he also talked about octave and then John Jacob Burgilius named several elements and give symbols for them and when periodic table was published by Dmitry Mandeleev he gave an important statement that physical and chemical properties of elements are periodic functions of their atomic weight but later Henry Mersey through extensive photoelectron spectroscopy he determined atomic number and several properties of elements and eventually he modified the understanding of periodic table giving importance to atomic number and thus it was modified as the periodic functions of atomic number that means physical and chemical properties of elements are periodic functions of their atomic number. Now we know that how valuable the atomic number is that is electronic configuration and number of electrons and once we know the electronic configuration and with basic understanding of periodic trends and periodic properties we should be able to deduce all properties and understanding the reactivity stability etc. And during the same time when Dmitry Mandeleev presented his periodic table Julius Lothar Meyer from Germany was almost ready even one year before but somehow he did not publish it does not mean that he did not contribute his contributions are very significant and due to some reason he did not publish his periodic table that was very similar to what Dmitry Mandeleev presented and however we can see in even in NCRT books Lothar Meyer's plot about trends among transfer elements and of course Glenn Sieberg and Yuri Agenison were given distinction by naming elements with atomic number 207 and 218 after their name and in fact when element with atomic number 207 was named as Sieborgium Glenn T. Sieberg was extremely happy and he said that it is more than this honor of naming element of his name is more than getting a Nobel Prize. In fact these are the only two people whose names were considered for two elements when they were alive and of course one should understand the trends in atomic radii or melting point, boiling point, ionization energy and try to analyze to understand the properties how it varies within the group and also along the period when you start filling from D0 to D10 electronic configuration. For example you can see why Magnus group shows decrease in melting point all these things are clearly spelled out in my lecture please give importance and attention to these things so that you can understand the behavior in a better way. It is very appropriate to remember Alfred Werner for his coordination theory again when he started working on coordination compounds of cobalt octahedral cobalt compounds and square panner complexes of platinum there were no support of any analytical or spectroscopic tools and not only that one even atomic model atomic structure was not known and even electrons were not known in fact he proposed his coordination theory in 1893 but electrons were discovered much later by JJ Thompson in 1896. However he did not leave any stone unturned with his synthetic ability and analytical thinking he could make all possible isomers and also he brought isomeration concept and also he said why coordination number 6 would prefer octahedral geometry when you have coordination number 4 why Saturn molecules prefer square panner geometry why Saturn molecules prefer tetrahedral geometry all those things very nicely he analyzed and he eventually he proposed his coordination theory during his time his contemporary who had more inclination towards organic chemistry Jorgensen was always opposing and ridiculing discoveries of Alfred Werner because he was proposing a theory called chain theory and always telling that no coordination compound can have coordination number more than 3 and also I showed you how the bonding was shown in octahedral compounds of having different number of ammonia and chloride in his chain theory and chain theory was a total flaw but however he was also ridiculing coordination theory proposed by Alfred Werner but in fact it even went to personal abusing level but however he tolerated all those things and he quietly worked hard and eventually he made everything very clear and then eventually he got the Nobel Prize in 1930 and one more thing we should remember is about making an optically active coordination compound having no carbon atoms in fact Jorgensen was again telling one cannot make any optically active compound without having a chiral carbon center so with this what he did was he made this compound you can see there are no carbon atoms only O and N are coordinating he made this chiral compound he isolated both the enantiomers and then for his painstaking work and of recognizing this synthesis he was given Nobel Prize in 1930. So now we know most of the concepts still were using whether it is reaction mechanism or whether it is isomerism or whether it is understanding bonding concepts and of course crystal field theory is a very nice theory to explain bonding among coordination compounds to an extent organometallic compounds but there is also flaw was there and nevertheless it is a fantastic bonding concept and it can explain many things only thing is it does not consider covalency in the bonding metal to ligand bonding. However the best from wellness bond theory that is mixing of orbitals that is hybridization and then crystal field theory about splitting of the orbitals were nicely imbibed into molecular orbital theory to come up with ligand field theory. Now ligand field theory having all these very good ingredients from these bonding concepts can literally explain everything including reactivity stability and all those things. So this is how coordination chemistry progressed from Werner's coordination theory and also in between ISO told you earlier attempts to explain bonding using electro neutrality principle and Keppert's model for using via CPR theory to explain bonding all those things. And of course crystal field theory is very very important in order to understand and to write crystal field splitting diagrams one should remember two things one is relative orientation of the orbitals with respect to Cartesian coordinates and also the direction of approach of ligand towards metal in different geometries. Once we know these things understanding and writing crystal field splitting diagram for any geometry would be rather easy okay. So again to emphasize I am showing you the orientation of orbitals and also the planes we come across x y plane and then x z plane and z y plane. So now if you arrange all the orbitals and if you place ligands coming from different directions to establish a certain geometry we can know now because of this interaction what would happen to the energy and how the degeneracy is destroyed and how they are arranged. Once we know understanding rest of chemistry would be rather easy this is for dx y square and this is for dx y and then this is for dx z and this is for dy z. Once we know these things and also we know the geometry of our ligand field we can write crystal field splitting very easily for any given geometry. So now this is very very important spectrochemical series and spectrochemical series the position of ligands in spectrochemical series can be checked by looking into UV visible spectroscopy of complexes having these ligands. But on the other hand why a given ligand has taken or occupied a position somewhere here or here in the spectrochemical series cannot be explained by CFSC. CFSC can also only tell you where it is positioned but why it is positioned you cannot tell for that one one has to understand the nature of the ligands and what kind of electrons they have and where in the donor orbits are located or whether it has donor and acceptor properties or it has only donor property or it has both donor and acceptor properties. So in that context these pictures are very very significant. So here you can see I have written three diagrams again I am emphasizing we talk about pure sigma donor ligands in that case CFSC would remain like this and when we consider a sigma donor and pi donor ligands for example low laying filled sigma donor and low laying filled pi donor orbits are there in that case what would happen because of overlapping and generation of molecular orbitals the CFSC drops significantly that is the reason these ligands are called as weak field ligands. So the CFSC does not tell why they are weak field ligands because of the these two here low laying sigma filled sigma orbits and filled pi orbits having low energy compared to metal T2g would signify why they are weaker ligands. And similarly if we look into sigma donor and pi acceptor low energy filled sigma orbits are there high energy empty pi orbits are there in this case what happens because of their overlapping the CFSC in this fashion CFSC increases. So that means we should remember this one the classification of ligands should be based on these three that is two sigma donors and sigma donors and pi donors and sigma donors and pi acceptors. Once we know those things understanding chemistry and utilizing them in some application would be very easy. Now let us look into M O diagram for square panner complex if we just look into CFSC crystal field splitting here this is the gap that determines CFSC among square panner complex and again here if you just look into it this is the gap that we call it as frontier orbits I would say and this is HOMO and LUMO gap here and if you consider here 8 electrons 8 electrons are filled here so up to here 8 electrons are filled and then this will be HOMO and this will become LUMO. So that means by extending this one considering this crystal field splitting diagram and then considering the what kind of hybridization one can anticipate for square panner complexes getting information from VBT and then we consider these two and put into molecular orbit theory this ligand field theory comes and it can explain very nicely all those things and also I have given Mullikan symbols for various d orbitals just go through it and just look into it and try to understand. So now one more M O diagram I have given here for nickel tetracharbonyl you can see here nickel tetracharbonyl this lone pairs are placed here higher in energy among all and this one essentially should go to the metal sp3 orbitals to establish metal to ligand bond metal to nickel to carbonyl bond but however if you just look into this diagram they are not participating there is no overlapping of this one with S and P because they are too high in energy so that means you should know the fact that NiCO4 does not have sigma or bonding at all that means how this molecule is formed it is because of back bonding only you can see here so these T2G electrons are getting placed in the molecular or generated by pi star and T2G of metal so that means only the back bonding from nickel to carbonyl oxide anti bonding orbital is responsible for the existence of NiCO4 so that is the reason they are very unstable and also they are volatile whereas in case of metal carbonyl such as chromium hexacarbonyl tungsten or iron pentacarbonyl we have seen the participation of both sigma as well as pi orbitals in fact all metal hexacarbonyls are stable and solids with having moderate stability so now these four diagrams will tell you non classical ligands having sigma under and pi acceptor capabilities carbon monoxide and phosphines and also fissure carbene and n heterocyclic carbene all of them are non classical ligands having sigma donor and pi acceptor capabilities and of course there is relative difference in their donor and acceptor properties carbon monoxide may be very good sigma donor and also good pi acceptor no doubt but phosphines can also compete well with carbon monoxide in terms of their sigma donor ability and pi acceptor ability but in contrast to carbon monoxide we can vary these things we can vary these properties to an extent that it can even perform as a better pi acceptor compared to carbon monoxide when we have electron with drying groups or electro negative substituents on phosphorus that means we should know that electronically we can tune the phosphines to make them better than carbon monoxide for example if you put more electrons releasing groups on phosphorus it can be a good sigma donor and a poor pi acceptor on the other hand if you put electron with drying groups on phosphorus it can become poor sigma donor but very good pi acceptor so this kind of flexibility in its synthesis you cannot come across in carbon monoxide that is the reason the tuning is very easy and hence phosphines are very popular when we use metal complexes containing phosphines as catalysts in homogeneous catalysis for several organic transformations and similarly fissure carbines also there here also back bonding is there back bonding one can anticipate in the same way as carbon monoxide but here already we have lone pairs within the ligand as a result what happens this intra back bonding is more facile compared to inter back bonding that is from metal to ligand as a result what happens they become poor pi acceptors so this also you can call this as intra back bonding also you can call it as negative hyper conjugation same thing is to in case of n heterocyclic carbines also you should remember n also has a lone pair this lone pair can also go to the pi star of carbine as a result what happens they are also poor pi acceptors but they are good sigma donors so this is how you can compare these non classical ligands in terms of their donor and acceptor properties and then 18 electron rule is very important it is not a must to have a stable complex because many square pen are complexes with 16 electron are stable for example rhodium 1 iridium 1 and also all d8 system nickel 2 palladium 2 platinum 2 gold 1 all those things but here 8 electron counting 8 electron give some idea about their possible utility in some reactions and other things for example if they are 18 electron complexes you cannot use them for oxidative addition reaction or catalysts prior to that one what happens you have to get it off a couple of ligands or you have to remove some electrons so that it is ready for oxidative addition reactions and also it is very interesting to count electrons so for the reason I have taken this interesting molecule again we have here rhodium and iron and we have 5 carbonyl groups are there and 1 C7 H8 is there cyclohexate triene is there and that I have shown here so now let us see how it satisfies 18 electron rule first you should think of a formal rhodium to iron bond and also why I have put more carbon monoxide ligands on iron is because it has less electrons compared to rhodium it is a d7 S2 system whereas d6 S2 system so now 2 are there and one rhodium rhodium rhodium iron bond is there and now you should place in such a way that both of them would be having 18 electrons it is 17 electron so that this comes through one comes through rhodium iron metal metal bonding so this is how you can show eta 4 and eta 3 so this is again very interesting so like that many examples I have discussed go through it and also you can find lot of examples in textbooks try to solve them or best thing is every time you come across a matter complex try to count electrons to see whether it satisfies 18 electron rule or not so now it is about metal to metal multiple bonding this is very important with square panor complexes and having eclipsed geometry of course I have discussed it in length how they identified first in the rhenium complexes in the group of FA cotton when he was in MIT and now we know that how to explain the bonding up to 5 bonds not 4 bonds like quadruple bond up to 5 bonds are possible between 2 metal centers and then how you know the bond formation takes place can be readily explained using even molecular picture like this and here you should remember the fact that dz square and for this one what happens to metal complexes having square panor geometry should be eclipsed to each other and in this case if you just ask me yx dx square y square is not used because that is already used for the formation of metal to ligand bond if you recall dsp 2 hybridization from wellness bond theory so that means 4 orbitals are left here dz square dyz dxz and dxy now one can comfortably use from 2 metals to establish metal metal bond like this so this one is a sigma bond head on collision dz square dz square and dxz is like something like this and then dyz is something like this they are degenerate they can accommodate 2 electrons each and then we have dx y is there so this is a weak interaction something like this so one is this dz square one is dxz another dyz and then this is dxy dxy will be like this so now that is called delta bonding between them weakest two overlapping among orbitals what would happen to the number of electrons one have d1 one electron is there so then you can have one electron from each results in sigma 2 double bond 3 triple bond and 4 quadruple bond if you have 5 again we start filling anti-bonding orbitals so triple bond d6 double bond d7 single bond and d8 everything is filled so bond already 0 you cannot see no formal metal metal bond can be seen in d8 system you can see that one again I am showing you here d4 system how electron filling takes place in this order to see quadruple bonding so all d4 system is square panel geometry have possibility of showing quadruple bonding okay so now it is even possible to have 5 bonds that is quintuple bonding for that one what happens you need one more orbital other orbital possible orbit is dx minus y square for that one you should ensure that metal has not utilized this dx square y square for establishment of metal to ligand bonding in that case we have to go for further lower coordination number for example if you take here in this case it has not used any of the d orbits for making metal-metal bonding let us assume it has SP orbits are there these 2 SP orbits have used this one here and this one so now all the 5 orbits are left and it also has 5 electrons in d orbitals now dx y and dx y square at an angle of 45 degree are degenerate similar to dx z and dy z so you can have here 10 electrons so bond order is 5 so this is how you can explain very nicely using m o diagram quintuple bonding as well I have given quite a few examples again go through it and in case if there is a problem always you can write to me and this is important about hydrogen bonding to metal in fact it is very significant h to h h bond is quite strong and it is endothermic it is not easy to break as I mentioned if you take any unsaturated hydrocarbon and put high pressure hydrogen into it and in a closed vessel and if you try to heat it for several hours even hydrogenation happens with very very low conversion about 5 percent or 6 percent but on the other hand you add a metal complex so that can happen even at room temperatures why that happens because how very nicely it drift the electrons present between hatch through sigma bonding and also it pushes its own electrons through back bonding to sigma star so that means you are taking away bonded electrons and you are pushing electrons to the anti-bonding as a result what happens h h bond becomes weaker and then earlier it is a eta one bonding it becomes what happens very nicely it adds oxidatively to form 2 m h bonds so this this is very very significant because of this property metal complexes have been extensively used in organic transformation in homogeneous catalysis so whether it is a h h bond whether it is ch bond whether a cc bond or any other hetero atom to hydrogen bond they can do very conveniently as a result we come across the application of these complexes in many organic transformations of course still we have not succeeded or achieved to use in industrial scale the cc bond breaking for that one one has to make very very ideal compound where it is highly electron deficient and the ligands are of poor sigma donor and coordination low coordination is there in that case what happens it should be ready to grab literally anything that comes on its way to expand its coordination number in this case probably let us say if you put octane it can go very nicely and it can break into 2 butyl groups if that happens with base metals such as iron cobalt nickel or something one can make fortune out of it so there is enormous enormous scope to activate cc bond using metal complexes but with cloverly designed ligands and of course here I showed why NO plus is stable because if you once get rid of this one electron from pi star it will be having bond order of 3 that is the reason it would be more stable and also here I am showing you now once this electron is gone and now NO plus is there this can also behave very similar to other non classical ligands and act as pi acceptor ligand you can see here I have shown sigma donation as well as pi acceptor so NO can also be used as a pi acceptor ligand and some metal complexes we can see these things. And when we talk about phosphines I mentioned about electronic properties and steric properties are very important the steric influence the magnitude of steric influence can be measured using Torlman's cone angle what happens here you take the ligand make a bond to the metal with a distance of average distance of 2.28 and strong units of 228 picometer now imagine a conical surface at the metal that encloses the van der Waals surfaces of ligand substituents over all possible rotational orientations how this controls the steric attributes you can see by looking into the cone angles shown by different ligands having different substituents as bulkiness of the phosphorous substituents increases cone angle increases in that case what happens you can have because of steric congestion you can stabilize metal with the fewer ligands in that case what happens we are automatically we are generating a low coordinated system having less electrons in that case what happens these compounds are electron deficient and also co-ordinatively unsaturated as a result they can readily undergo oxidative addition for example if you take compound here phosphines four phosphines are there in tetrahedral 18 electron system is there the moment you put into solution because of steric congestion two ligands go out dissociation would be very easy and now let us look into the metal to halide bonds in metal to halide bonds also I discussed it in length about how certain ligands certain metal complexes have tetrameric structure and some of them have dimeric structure take even aluminum itself aluminum fluoride if you look into it it has a tetrameric structure like this whereas aluminum chloride has a dimeric structure like that AlCl3 become Al2Cl6 Al2Cl6 it is not aluminum actually it is a metal complex here it is a transfer metal complex but nevertheless the reason is the smaller size when the smaller size is there because of more electrons are there if assumes a fluoride assumes a bent structure when it is bridging to two metal centers in the bent structure what happens two cations come very close to it they repel as a result what happens they would try to have a linear geometry in that case if you want to see association that has to be minimum of tetrameric or in some cases trimeric also we come across the preferred one is tetrameric hence if you look into CuF22 plus or CrF22 plus although it looks like they have linear geometry it is not linear geometry the composition of copper to fluorine is 1 is to 2 and but they have octahedral geometry something like this and then oxide tradition is very important and I also discussed in length about different possible mechanisms we have 3 bond concentrated addition is there and nucleophilic substitution reactions are there and also radical mechanism also there and also I discussed all those things and you can see polar solvents are going to the transpositions whereas nonpolar bonds are going to the cis positions so we can see clearly distinguish between concentrated addition and also nucleophilic oxidative addition and also I discussed it about a stereochemical consequences also and then trans effect is very important when it comes to substitution reactions in square panner complexes what one should remember is in a given complex electron attracting ligands are epical and electron repelling groups are trigonal planar so when the incoming ligand is there a pair having trans influencing group and a group trans to it and the incoming plane will be trigonal planar and other two will be axial and in this case you have to identify which is electron attracting group and which is repelling group and accordingly if you generate the intermediate the moment you generate intermediate you will come to know the conformation what kind of conformation we are going to get at the end of substitution is completed and of course in case of octahedral complexes I did mention about substitution how it happens incoming ligands can come to come on the same side of the group or they can come on the opposite side and accordingly what happens you can see how that influences the formation of cis isomer or trans isomer and also I also discussed about stereochemical changes the stereochemical consequences in a substitution reaction on octahedral complexes and when it comes to redox reactions we have two type of inner sphere coordination and outer sphere coordination and in case of inner sphere coordination theory we need a bridging ligand and this is how the intermediate would be there and you can see here once electron transfer is over this also moves towards the other one that that is getting oxidized and then of course in outer sphere mechanism we have to keep frankendon principle in mind and now we can see here t2g eg he is there in this one is a high spin complex where it is a low spin complex before electron is transferred both of them should have a optimum bond length and bond angle and now you can call it as intermediate stage where electrons can run smoothly so that this also does not disobey frankendon principle once the electron transfer takes place they will revert back to different one so this becomes high spin and this becomes low spin and then spectral interpretation I made an effort to make you familiar with you know UV visible spectroscopy and also NMR spectroscopy to an extent IS spectroscopy so that you can characterize those compounds are if you come across some spectra you should be able to interpret and various methods and how we can arrive at the structure all those things I have shown in this chart and of course angular meant quantum number and spin angular momentum how they interact I have shown here very nicely LS coupling and LS coupling is also very important when we talk about UV visible spectroscopy and also in when we look into magnetic properties and then of course I classified all de-electronic configuration to four groups here there is a significance why we have why I have done like this here you can see one the charge transfer transitions if the compounds are color it is because of charge transfer in this one ligand to metal in this one metal to ligand and in these cases we have one electron one less than half field one more than half field and one less than completely filled here in the same way we have two electrons and two less than half field two more than half field and two less than completely filled so these complexes show invariably one dd transition here they show invariably three dd transition of course d5 is spin fermentant and leopard fermentant and compounds are weakly colored or pale colored compounds and of course all those things I discussed in depth and this equation is very important when when we talk about NMR and using this equation we can literally explain anything about NMR transition and other things and what one should remember is if you look into spin selection rule delta s equals 0 so that means basically a electron with upward spin should go like upward spin only whereas in case of NMR its plus or minus 1 the flipping of nuclear spin I mentioned so electron with upward spin should go to exact state to be having a lower spin alpha becomes beta or something like that so this is very important and you can see here in this diagram precision results in the flipping this is through radio frequency applied in a direction perpendicular to the magnetic field you can see clearly here so you should remember these things and then magnetism I did not have time to discuss about magnetism of course you can find these things in standard books pretty easy and we use two equations and of course here we are using s is the summation of number of unpaid electrons here for example s if one electron is there s equals half two electrons are there one and then three electrons 3 by 2 it goes like that here n is number of unpaid electrons both are essentially same and if we can also use this one where of course you know already how to find out L as well as s you can find out mu from this equation as well or in case of lanthanide and actinides we have to find out G G is 2.0023 and if in some cases only whenever we come across spin only treatment we can always consider G as 2 the splitting of orbit levels is larger relative to KT that is Boltzmann constant then one can use this equation here G into square root of J into J plus 1 G can be calculated provided we find out from electronic configuration what is s and what is L and of course J one can also use value L plus or minus s depending upon whether the orbital is completely less than half field or more than half field if it is more than half half field we use L plus s if it is less than half field we use L minus s where we should consider you know orbital contribution we have to see they should be differentially especially T 2 G orbits in case of octahedral complexes and T 2 orbits in case of tetrahedral complexes should be differentially occupied in that case what happens you can see spin orbital contribution the moment you look into the electronic configuration you should be able to make out whether spin orbital contribution is coming or not for example in case of octahedral T 2 G 1, T 2 G 2, T 2 G 4 and T 2 G 5 have unsymmetrical filling in that case you come across orbital contribution and same thing is to in case of tetrahedral X Z of course here G should be removed because it does not have centrosymmetry it should be T 2 1, T 2 2, T 2 4 and T 2 5 for tetrahedral complexes and you can see here with 45 degree rotation DX Y can become DX minus Y square and similarly with 90 degree rotation DYZ can become DX Z. So, at last I dedicate this lecture series to my beloved parents my father and my mother one year before I submitted my thesis my father passed away and two years back my mother passed away and these people taught me high ethics in me and because of them what I am today. So, I dedicate this lecture series to my parents and also you should remember parents always work hard to make you better citizen and educated and they sacrifice everything. So, in that context always try to remember parents and of course in some cases what happens I have seen students parents force their children to opt for a particular topic in which the student a son or daughter is not interested in that case what happens you should try to convince them why you are interested in this one and why you are not interested in what they are imposing certainly you can convince them and you can take a right path and achieve greater success and I am sure I had conveyed some chemistry through these 60 lectures and I have put lot of effort you should remember lot of effort I have put in generating some of the slides to convincingly teach some chemistry and when you understand the chemistry behind these 60 lectures and you know pass exams with good grades and if you are at the end if you think that yes I got something out of this course I will be very happy and in case if you have any problem you can always write to me and later when you achieve higher success and in case if you think that this course has contributed to your improvement your knowledge and when you achieve greater success and achieve something and make a mark and if you just send me a mail I will be the happiest person and that brings millions worth happiness to me. So, with this I will show all very very best and God bless you all enjoy chemistry thank you so much learning never stops even now I am learning I can say without any hesitation I am a good student of learning underlying good student of learning because I do not claim anytime that I am a teacher because I have to learn a lot even now and that means no matter what you do learning should never stop. So, learning should continue so that we can have more and more knowledge and when we have enough knowledge it is our sincere duty to dissipate it to others thank you once again.


What's up everybody, Jason here for jazbyzcasebrakes.com. We just sold out 2022 Panini National Treasures Football. One box break, pick a team's number one, and here we go guys. So this is coming from a fresh case. What's up Bernie? All right, so, here are the customers that are part of the break guys. Everybody else of course got in through a R&B. So this one right here guys, for all the big teams guys. All right, National Treasures Football. All right, so we got four boxes guys. One, two, three, four. See if it lands on a one through four. Five, no. Six, no. Three, there we go, finally. Third time's the charm too. All right, three, here we go. What's that guys, that's funny, what a one. Wow, well everybody's, everybody has different, everybody has different rules. So that worked out in your favorites. Yeah, cause someone asked like, oh, if you have the zero spot, do you automatically get everything? You know, do you automatically get all the non numbered stuff and everything and we're like, no, we actually just give that out to everybody, but everybody has a different. It's cause really honestly, the only reason why we don't give it to number one and it's kind of just, it kind of just makes sense, but see knowing publicly to one to one, okay, you do know the certain number is going to be one of one. So I can see why people would do that, but then if it's a redemption and no one knows the other number, right? You know it's out of 25, but it could be literally any number. So I just think to make it safe or to make it all easier for everybody, it's just more like just, you know, keep it all the same. Everybody gets the zero gets all the redemptions no matter what it is. But yeah, good for you, man. That's actually just pretty awesome. All right, for the Jets, here we go. That is Joe Namath, 64 out of 99. That's, we're going to go to the Jets R&B, 64. When is Steve Herrick? Then Alvin Kamar, same to you. All right, Devon Cook, 1099. Minnesota Vikings, Eric. And we got a Detroit Lions, Jamal Williams, Relic. 37 out of 49. And that's Detroit Lions R&B, seven, which will be going to Viviana. And we have a Trey Lance. That is 10 out of 25, two color patch there. 49ers with that one. And that's of course part of the R&B. Zero going to Viviana, last ball Mojo. What about that? Look at that, not a pretty autograph, but you got a pretty two color patch with Danny Gray, seven out of 10. Spot seven is going to Viviana. There you go. We have Thurman Thomas, patch autograph for the Bills. Bills is part of the R&B, 46 out of 99. Spot six is Allen. All right, a couple more guys. Dual autograph, how about rookie prime pairings? Romeo Dubs and Christian Watson, 14 out of 25. Green Bay Packers going to Ed. Hey, there you go, Ed. Haven't we been hit this for a while? There you go. All right, two more to go guys. We have a Brian Erlacher, 12 out of 49. Bear's going to Eric. There you go, Eric. Little Erlacher for you. And last but not least, we have ourselves a RPA. And it's actually a really good player, guys. It's to the Jersey number RPA. It's Aiden Hutchinson, three out of 97. Not a bad RPA, not a quarterback, but still not a bad RPA though, he is the top pick. And that is going to go to the random number block. And it's going to be spot three, which is Allen. There you go, man, congrats. Very, very nice. Solid box, actually. I know it wasn't a quarterback, but solid box there. Again, it is from a fresh case. There still could be a big RPA quarterback hiding in there, whether it's a picket, whether it's a purdy, Sam Howell, any one of those guys, but a solid little box here. I'll post another one up, guys, and we can start trimming down some teams. Get that done again, guys. So appreciate it. Jaspeys, casebreaks.com, guys. Thank you.


This study suggests that perceived social support can reduce the risk of stroke associated with anger expression among individuals with low levels of social support. This article was authored by Kazuhide Tezuka, Yasuhiko Kubota, Tetsuya Ohaira, and others.


I'm kind of letting the curls set in, but first we're gonna go to eyes because I have a feeling I'm gonna do some glitter. I don't really want the fallout all over my foundation so I'm just gonna zoom you in really quick. First I'm gonna be taking my Naked Skin Concealer and priming my eyelids. I'm not really sure if I should do a neutral brown or green or blue. I don't really know I have all these colors to choose from so if I'm kind of everywhere with my shadow colors just bear with me because I'm just kind of rolling with it. I didn't practice before. The only thing I did practice was the mermaid scales which looks so cool and I can't wait to show you guys because it's so easy to do. So first I'm just gonna go in with a neutral color. This one is from Makeup Geek. It's called Latte and I'm gonna be placing this all over the lid and in the crease. Then I'm taking Cocoa Bear and putting this in my lid and crease as well. Next I'm going to attempt to play with this green color called Envy. It's from Makeup Geek as well. It does have some shimmer in it so I don't really know what I'm gonna do it with it but think lid maybe? Yeah let's go with lid. Then make it as seamless as possible so blending the Cocoa Bear and the neutral browns into the green. Next I'm taking the Sapphire color from Morphe and I'm gonna see if this makes it pop more. I guess it didn't really do much to the color payoff. I think these colors are just a little bit dark and I kind of wanted something a little bit more bright but I think the gradient will look really cool. I'm going back in with Envy and putting that on the inner half of my lid. I actually really like this gradient. I didn't think I would like it that much so really happy with the way it's turning out. And now what I'm just gonna do is blend it all out with Cocoa Bear. So this is what my eyes look right now and I think I kind of want to add some gold to it. I don't really know what kind of gold but I just kind of want to make it look a little bit more rustic. So I'm gonna take this color Soakiesh from ColourPop. It's kind of like that rustic-y mermaid look. It's really cool I don't even know I had this so that worked out. And I'm gonna apply this with my finger and just put it on the lid. Then I'm just gonna blend that and make it a little bit more seamless. I'm definitely gonna clean it up a little bit when I put my foundation on but this is what it looks like right now. It kind of turned into like more of a smoky black eye for some reason but I'm gonna just roll with it.  So I will see you guys in a second. So I use this stencil that I got from CVS and you can also buy this on Amazon. I will link it down below. Make sure to get a stencil that is also a sticker so that it can lay on your face flat while you paint it. So next I just took some acrylic paint and I know this probably isn't the best for my skin but I didn't really want to go out and buy face paint since I had so much of this laying around. So I used a blue one and obviously you can use any mermaid type color like a purple, a green, a teal. I just painted the stencil kind of framing my eye and then going right above my contour. The next step is to add glitter. I just took a brush and kind of patted it in on the wet paint and make sure it's wet so it can stick so you don't have to use any other glue. Next to give it that rustic mermaid look I'm going to be taking Nillionaire by Colourpop and applying this with my finger on top of the paint and glitter. Adding a gold on top really gives it dimension and I also recommend putting some gold where it's not painted blue. I went back in after this and just put some plain gold scales right beneath the blue paint and it looks so cool and I also put some plain gold scales on my collarbone. Make sure you're not as impatient as I am and don't rip it off while it's still wet because the stencil might take it off like it did to me so definitely wait till it's completely dry. So I wanted to put some scales on the other side of my face too but I didn't want it to be super symmetric so I put most of the scales on top of my brow instead of doing it on my cheekbone. This is the finished look and I hope you guys will recreate this for this Halloween and if you do definitely send me pictures on Twitter or Instagram tag me on any photos that you do this makeup look for because I would love to see them. So for my outfit I wore these spanks which I got offline. I will link them down below as well. They just have that scaly mermaid print and then for my top I actually DIYed this and I put the seashells on it with just acrylic paint and then put some glitter on top of it and I thought it turned out really cute but that's it of this mermaid tutorial. I hope you guys enjoyed it and I will see you guys in my next video. Bye guys!


One of my favorite things about her, whatever, is those are bumpers for her.  This is a modified form of chaff and redirect, because she doesn't have a real plan where the chaff is going to take her and something to throw out that you'll pick up and run with. She proves that, because at one point, the woman asks her a question and she's like, oh, oh, and she stammers to answer it. So she's not chaffing and redirecting. She's chaffing until she gets the opportunity to say, this guy needed to be killed, is basically what she's saying. When she does whatever, blah, blah, blah, she's avoiding something significant here in my opinion.


So go ahead and get started. So it's my great pleasure today to introduce Annabarie Pyle, who's giving this year's David Green Lecture in Enzymology. And I just thought I would give a little background on David Green. So this particular lecture is supported by the David E. Green Memorial Lecture Fund. And there's one of three named lectures we have here in the biochemistry department. And so David Green, like Anna, began his independent career at Columbia University. He then saw the light in 1948 and moved here to Madison, to help found the Enzyme Institute, so the Institute for Enzyme Research. And while here, he was extremely prolific. So the number I found on the internet, which has to be true, on the internet, says he was associated with 700 articles that were published over four decades here in Madison. And he really made fundamental contributions to our understanding of oxidative less formation, beta oxidation, and many other enzymatic reactions that occur in the cell, particularly in the mitochondria. And he also established the Enzyme Institute as really a foremost center for mechanistic enzymology and helped recruit many people in that area, including Lardy, Cleveland, and Piranha, and many others. So he also had a nice quote I found that I think is particularly appropriate Piranha's research in maybe the field of RNA splicing in general. So at age 27, he published an essay titled Reconstruction of the Chemical Events in Living Cells. And in that essay, he wrote, the mastery of a particular machine requires not only a knowledge of the component parts, but also the practical ability to take the machine to pieces and reconstruct the original, which I found to be a really nice quote for what it means to understand something in mechanistic and molecular detail. And so today, I can't think of a more fitting lecture than Annemarie Piles' work on RNA splicing. So it's very well known for studies on the mechanism of group 2 introns splicing, kind of all of those, the molecular details of catalysis, how these ribosomes hold, and their structure of these ribosomes. In addition, she's really made really nice contributions to her understanding of RNA helicases and how these functions inside the cell in a variety of ways. She's currently at Yale, and she's also an HGMI investigator who she's been since 1997. And she holds the William Edward Gilbert chair of molecular and cell and developmental biology. She's also a professor of chemistry at Yale. So with that, I'll be able to honor and thank her for her degree to come to this lecture. Thank you, Erin. Well, it's an honor to be asked to give the green lecture. It's also an honor to be asked by Erin, who has just done such amazing work in such a short time. And it's also super fun to come and see all of my many friends here. You know, as part of the already society community, I spend a lot of time here organizing meetings and coming to meetings. And this is sort of a second home. So anyway, it's nice to be here. So in part because this is the green lecture, I'm going to be telling you about splicing today. And I'm going to try to do as much emphasis on sort of enzymology and how these functions as splicing enzymes as possible. And so for the undergrads that are present, I also am going to give a bit of an introduction to splicing and mechanism of ribozymes. So for those of you who think about splicing all the time, please forgive me. And we'll ultimately get to something that hopefully you don't find interesting, too. OK, so right here is my favorite splicing machine. And this has been the focus of my work. The group two is self-splicing and trans-family. We'll be learning a lot more about that. But you know, it looks pretty complicated, and it is. So we're going to start with something a lot more simple to begin. One thing that needs to be said to really understand why we're talking about any of this today is that RNA is not functional until it's spliced. The central dogma created some major ribbing. DNA doesn't just go to RNA, it goes to protein. The RNA has to be cut and patched in various ways and modified in various ways before it's functional. So here is what splicing looks like for an RNA with one intron. An epsilon is defined as a piece of RNA that encodes something functional, like a breaking sequence. And often, two exons are subdivided by a region of RNA that doesn't actually encode for something important or may have secondary functions. And it's called the intron. And through the splicing reaction, this piece of RNA is removed, usually in a lariat form, I'll explain in a minute. And the exons are spliced together in a simple way. OK, so that sounds very easy. Although it's important to see that this is a two-step reaction that we'll look through that in a minute. But life is not that smartly to have lots of introns. It's going to look something like this, where you have various different exons that have different modules of, let's say, a breaking sequence. And the boundaries between these exons have to be spliced together to create this sort of multicolored pulse that is now ready for function. The why is splicing important. It's really important to you, because most of your genes are in this category. You have an on-and-average eight or nine introns in all of your genes. So splicing is a huge part of your gene expression situation. And it gets worse. This is what it looks like when every one of these exons is included. And you get a single-arge product that encodes, let's say, one possible protein product. However, life is not that simple. In us and in other complex organisms, exons are spliced in various combinations. So maybe the red guy will splice to the yellow guy and leave out the orange one, et cetera, here. It's giving you one of many combinatorial possibilities in gene expression. So in our tissues, our tissues all undergo what's called alternative splicing with different combinations of exons. We express different kinds of spliced products at different developmental stages. When we're early embryos, we make different combinations than when we're adults. And what this has done is that splicing has enabled eukaryotes to break the one gene, one protein barrier. So this has facilitated massive organismal complexity. So we can do a lot more with a lot less information through this process. So that is why splicing is important. So this is the first message I got, especially in use, but not exclusively in use. If somebody tells you that there are no introns in prokaryotes and in bacteria, they are not right. There are lots of group 1 and group 2 introns in bacteria. The second message I'd like to tell you is that RNA splicing machines are predominantly ribosomes. In other words, they're machines that are made of RNA. They're enzymes, but they're not substantially in their active sites composed of protein. So again, this kind of thing is catalyzed by RNA. So what are ribosomes? Ribosomes are RNA molecules that catalyze chemical reactions. They are true enzymes, although many of them undergo autocatalytic reactions with one turnover. Ribosomes come in many different flavors. And I like to group them into these different sort of flavor groups in the following way. There are big, giant ribosomes that mostly act to clean unlike a RNA and DAA. And they are basically two of them. There's a self-blessing group 1 intron. There's a ribosome that clips the termini off of your tRNA molecules when they're initially made. There's the group 2 self-splicing intron that we'll talk about. And now we know that the splice zone, which as we'll see is the big machine that processes with multiple turnover, all of your splice sites, is also going to fall into a category that shares a common enzymatic mechanism. We're going to talk about this sort of catalytic mechanism of this class in great detail. Now I'm going to just run through the other classes that people like to think about. We're not going to talk about them further in this discussion. There's another really big group of ribosomes. And they're all kind of tiny. So some of them are as small as 19 and as large as 80 or so. And these are often structured RNAs that are present in viruses, and viroids, and other contexts. And these are self-cleaning motifs that are conserved and fall into these families. They undergo an RNA reaction that's actually pretty simple, in which a general base deproved needs the 2-prime hydroxyl at the sysile linkage, the bond that needs to be cleaved, and gives you a site with phosphate at the end and release of a 5-prime hydroxyl. OK, that's these guys. So let's just say these guys have an easy task. They do a pretty easy reaction. Because as you know, this is sort of a natural degradation reaction of the RNA. As we'll see, these guys have a much more complicated molecule. The other big member of the ribosome family is the ribosome, in which RNA catalyzes peptidyl transfer. The heart of the ribosome is all RNA. And RNA is doing almost every aspect of the peptidyl transfer chemical reaction. And then something you may or may not know about is that there are DNA molecules that can catalyze, especially RNA and DNA cleavage and ligation. These have been developed mostly in a test tube in vitro through selection methods. But Ashley DNA could be a very good catalyst if you select for one that is targeted to a certain type of chemical bond. So those are sort of lurking in the background and they're sort of artificial. But they're interesting. OK, so having defined ribosomes as a whole, let's go back to talking about the group that we're going to focus on today, these big ribosomes. The large phosphodiesterase ribosomes catalyze this particular reaction in which an exogenous nucleophile, and by exogenous I mean it's not this guy, it's a nucleophile that comes in and does inline attack as a separate molecule or a separate region of the molecule, does inline attack on the phosphorous, giving you trigonal bifuramid structure of the intermediate. And so you get this kind of architecture that will require subsequent prognation of the leaving group, and you'll get a 5-prime phosphator linkage and a 3-prime hydroxyl. Now, everybody in this family of big ribosomes catalyzes this kind of reaction. And when I say nucleophile, it's always some kind of alcohol, either a 3-prime hydroxyl, 2-prime hydroxyl, or water. OK, so you'll see here, I circled two big red circles here, and if you can see the print, it says magnesium. These reactions are almost always metal-catalyzed, and this was discovered not through the structural biology done in my lab, although it was confirmed by that, but this was initially demonstrated through chemical biology methods that were pioneered by a guy named Joe Pichirilli, who's at the University of Chicago. And he basically proved the involvement of these metals in stabilization of the leaving group and of the nucleophile by doing this really creative experiment.  And it's called a metal ion specificity squad. And what he did before we had any of the structures I'm about to show you is that he substituted these potential ligands here, like these oxygens, with a soft ligand like sulfur. And he saw that when he did that, the reaction no longer proceeded in magnesium. Magnesium doesn't like to interact with sulfur. But if he dropped in a reasonable fraction of cadmium or zinc into the mix, he could rescue the reactivity up to wild-type levels. And in that way, he established interactions between metals in the leaving group, the nucleophile, and I'm not showing you here, but also some of the non-virgin phosphoryloxygens. So this was a very important experiment, and it established that enzymes of this family proceed through a two-metal ion mechanism. He did this with group two introns, which we'll talk about in detail, and with dysplasosome, which we'll also discuss. And this was done earlier by Scott Strobel and others and Joe with group one introns, and as we'll see also with RNA-SP. Now, what I'm gonna do right now is jump way ahead to very recent structural biology, which says that these predictions were correct. Here I'm showing you a zoom-in of the active site with a bunch of our favorite large ribosomes, group two introns, group one RNA-SP. And what's hard to see here is the RNA that sits, that sort of presents the sisile linkage. But you'll see that in each of these cases, there are two metal ions that are four extremes apart that are in the ideal position to provide a two-metal ion mechanism, much as you see in the polymerase, plenofragment, and other protein enzymes. And in fact, for a parallel, I'm showing you here, RNA's H, which also presents two metal ions in the appropriate position to, or to cleave the sisile bond. So the reason I show you this is that these are RNA enzymes, and this is a protein enzyme, but they're all doing the same thing. And they're all basically utilizing a scaffold with a different kind of chemical composition to accomplish the same task of RNA cleavage. Okay, so this is the reaction that they all share. And so, now we're gonna funnel down our attention a little more, and we're gonna focus on group two introns in the splicing zone. And the reason I'm gonna do this is that these two types of large ribosiline are the ones that do pre-MRNA splicing. So the splicing of messages to make mature proteins. Group one introns are important. They're important in a variety of bacteria and certain primitive eukaryotes. They're often important in ribosomal RNA splicing. These guys are what we need to do pre-MRNA splicing. So we're gonna focus on those. Okay, so a little bit more about these. Group two introns are auto-catalytic in the sense that they form a structure that folds and presents a single special backbone linkage, a single two-prime hydroxyl group that serves as the nucleotide in the first step of splicing. That releases the three-prime hydroxyl group of the five-prime axon. And in the second step, that attacks over here at the three-prime splicing, ligating the axons and releasing the variant intron. Now we know that very similar products are produced when we undergo nuclear splicing with a big machine called the splice zone. And this is what processes our RNAs in the eukaryotic nucleus. The splice zone is a little more complicated, as you can see. It is a group of conserved RNAs, and not shown here is a host of auxiliary proteins that are also important in this process. This is a unibole molecular reaction, it's a one-shot reaction, whereas this is occurring with multiple turnovers. The splice zone actually acts as a true enzyme. It operates on the set of splice sites and then it gets up, reassembles, and that's on another set, okay? So for a long time because they had similar splice sites and similar products, it was thought that they might share an evolutionary heritage. But we all questioned whether or not that was true. It also implied that the heart of the splice zone might be arrived as much like the heart of the group 2 intron. This was all speculation, and I actually didn't really engage in it very much. I just thought group 2 introns were really interesting. And I knew that it would have very cool structures because they have lots of long-range interactions that I can see by cross-linking and lots of other things that suggested that their shapes were really elaborate and different than anything we've ever seen before. So I decided to focus on those and to be agnostic about the connection of the splice zone. And anyway, I spent about 20 years studying these as enzymes, trying to figure out their structures, and more recently getting very high-resolution structures to figure out how they work. So what's the story with group 2 introns? Once again, like any form of pre-already splicing, they undergo the same two-step reaction I showed you before, where a specialized 2-prime hydroxyl group at one point in the intron attacks. You get step one, the least hilarious is step two. Now you'll notice there's a little more detail in this diagram. I'm showing you that the splicing reaction not only goes forward, it can go very well with reverse. So this reverse reaction is an important thing to keep in mind because after you excise a free group 2 intron, it's still a very reactive enzyme. And it can attack pieces of RNA or DNA and insert itself back in them through the reverse reaction. And this becomes important when you see that group 2 introns have another life. They're not just self-splicing the RNAs, they're retro-elements. And they played a big role in the dispersal of introns throughout all of terrestrial genomes. Okay, so what do group 2 introns look like? Their secondary structures show them divided into about six domains and this is largely one ounce of true over many years. They do fall in three major families, which we'll see in a while, but those all show the following things. The first domain to be transcribed after the 5-prime exon is domain one, which is the largest domain. Domain two plays a small role in the architecture. Domain three plays a moderate role. Domain four does nothing for active site architecture, but it does encode an open reading phrase. So stuck onto the periphery of this ribosome, you can have a huge gene encoded here that gets translated and the translated protein can come back and influence the splicing reaction. It also becomes the site of the protein binding. Now the most important part on a group 2 intron is domain five. It's also the only part that is highly conserved in sequence. So the thing that really confounded the initial people who discovered group 2 introns splicing were these very innovative people, was that it was impossible to learn about their architecture using just plain old genetics or by staring at sequence. There just wasn't enough conservation or phylogenetic co-variation to understand them. Almost all the conservation was here and as we'll see in our link right here. But it was very clear that after domain five, there was a domain six that contained a bulged adenosine that would attack the phyprime splicing and you'd get the first step of splicing, this would attack the second step of splicing and you'd have a laryed intron much like you get from splices on the processing. Okay, and these little sequences I've highlighted here are regions of the phyprime axon that are recognized through base pairing with regions of domain one. So these guys base pair, these guys base pair and you can change them to anything you like and preserve the structure. Okay, so that's how group 2 introns are organized overall. Now, one more slide about what they do.  You don't really want a free group 2 intron working around because they do the following thing. After they're in size and if their open reading frame is translated that resulting protein comes back and forms a revenue clear protein particle called a group 2 intron polo enzyme. So you have this RNA protein complex between the group 2 intron and it's encoded protein which is called a match brace. This complex then does an amazing reaction. The intron can recognize on DNA sequences that look a lot like its recognition sites within the intron and it can reverse splice into the sense strand of a homologous piece of DNA. And then the protein partner, it has a DNA and a nucleus domain and it can clip the anti-sense strand and then if that weren't enough amazing stuff that this protein does the biggest domain of this protein is a reverse transcriptase domain which then comes back, uses this as a primer and makes a complete DNA copy of the inserted RNA intron. So this is one of the ways that group 2 introns came to just find any genome that they entered and to proliferate within those genomes. There are certain organisms, eublina being one of them, that in which 30% of the genome is now group 2 intron. And that has had a big impact on how those organisms diversify and appropriate. So anyway, that is one of the other kinds of things group 2 introns do. Okay, so we studied both the splicing and reverse splicing reactions of group 2 introns for many years working out chemistry, enzymatic function. And we reached this point where we really couldn't get any further until we had high-resolution structural information on this system. So the field was really at a standstill. So if we wanted to really understand the chemical mechanism, how the RNA actually organized itself to be a ribosome, to be an enzyme-acrocyte, we needed high-resolution structural information. So we needed to go from this, which is the secondary structure of the intron we were working on, which is from the east, we're from Kavria. We often call these secondary structures roadkill maps because it basically looks like you're earning after you've run over it with a truck. It's completely flat. And so it definitely lacks functional information if you care about enzyme-acrocytes. If you add magnesium to basically the roadkill map, it'll fold up into its native tertiary structure. And this is the tertiary structure model of this particular intron. And it's very highly organized. And we spent the next 10 years or so studying how to sort of investigate the function of tertiary architecture. But first, we needed to get high-resolution structural information. So when I embarked on this, people said you'll never get a crystal structure that hardly is good. You should just give up now. So I actually, in parallel, did a lot of cross-linking, a lot of chemical probing, lots of other things, which was good because that data enabled me to later validate the crystal structures I got. But nonetheless, we ignored all these errors. And we spent a lot of time looking for group 2 introns that would fold at very low magnesium concentrations and that would be very, very stable. We finally identified one genome of eudacterium in oceanic bacillus agensis. And after we transcribed this, we folded it into a typical group 2 intron structure. And then we made many, many constructs. I should say the first construct that we made of the OI intron, when we put it in crystallization drops, actually crystallized pretty readily. When we looked at the diffraction, it was at 20 angstroms. But we were very happy. We were at the synchrotron, and the staff member of the synchrotron said, this is the ugliest diffraction pattern I've ever seen. This is the most beautiful diffraction pattern I've ever seen. So we were okay because your first iteration is just a start. When you want to solve an RNA crystal structure, then you actually do the application of the architecture to get better packing into a proof of resolution. So making lots of RNAs for structural biologies, a lot easier than making lots of proteins. So we don't have to over-express this. All we have to do is make a plasmid and use over-express T7 RNA polymerase just to make as much of the RNA as one. And it has to, never has to go in and be expressed in bacteria and in yeast or anything. So we made 127 different constructs of this guy, and we varied the lengths of various stems, loop sequences, and other things, and just kept crystallizing them and looking for which ones diffracted to highest resolution. Finally, number 87 crystallized to 3.1 instruments resolution. And we've since done better.  What you see before you draw the model, and I've actually drawn the model of the back one to help your eye follow it, but when you're doing RNA crystallography, your electron density map immediately jumps out to you as having a discrete RNA structure. So yeah, you can see these nice helices. You can automatically begin modeling pretty easily into this kind of data. So we've solved many of these structures. Our best crystals diffracted to 27 instruments resolution This was phased with heavy-added derivatives several times in several ways. We used a curvidium hexamine. We also used echerbium initially. Echerbium is binded to the catalytic medallion site, which is nice. Later on, as I'll tell you about, there are very key monovial land binding sites in this RNA, and they bind to potassium selectively, and we can look at those by substituting in with phthaline curvidium. We have very good, our free values. This led to a lot of new methods because RNA crystallography is kind of still early. And fortunately, our structures agree with all the biochemical constraints that were kind of hard to find when we were doing a lot of brute force cross-lamping on the architecture. So we're pretty confident that it was a meaningful set of structures. Here's what it looks like. This is the oceanobusilus igansis domains one through five, which for this intron has always been able to get. Domain six is dynamic and we never observed it. Now, color coding here, I told you that domain one, the bioprimed most domain, folks first, and it's the largest domain. And you can see here, it almost forms what looks like a scaffold into which all the other domains dock. And we'll see actually that turns out to be true. The other domains fit into specific positions. The most important being the catalytic domain five, which contains the active site as we see. And you can see as this thing turns around, the active site left has the bioprimed splice site tucked into it that consists of phosphate. And as we'll see later, there are metals that surround us and participate in the cleavage reaction. But we'll get to that in a minute. To me, more than just the enzymology that we learned from the system, we began to learn a lot more about tertiary structural organization. What are the nuts and bolts by which RNAs hold themselves together through interactions that are not base pairing? So this shows you just a few of the many examples that we've learned just from that one structure. We learned that big junctions in RNA can be stabilized by all kinds of unusual interactions. This is a really cool interaction in which a loop region of one RNA contains sort of a slot for a base that's provided by this whole region of RNA that comes in and introduces this and interpolates it into the structure. And this is actually a conserved motif. And you can see that kind of holds the thing together like the way people used to make fine furniture where you'd actually have tiny and rude structure to it. Also, you see here stats of three-stranded and four-stranded structures. What I'm not showing you here is we don't really have time. There are lots of interactions that are mediated by the two crème microssal groups above the backbone. This is probably my favorite part. And Jim Keck was organizing his lecture around this today. This is reminiscent of what line is falling about what happened in DNA. So there's parts of this structure where loop regions interact with each other through a three-stranded motif that's pretty interesting. There are two helices that melt together through the internal loops in the following way. There's a duplex that comes up here and it flips out one of its bases and that base interacts with the third strand over here. The next base flips in and forms a canonical base pair. The next guy up interacts with the partner over here giving a zigzag appearance and having this strand actually participating in a three-stranded interaction. So line is wasn't totally gone. You can have RNA that's inside out if you do it in small doses like this. Anyway, so there were lots of really cool structural and I think that was what I was most excited about for a long time. Now we can skip over about 10 years worth of mechanistic work on the RNA folding pathway to just show you a movie that describes how the intron folds. So we actually have done a lot of a lot of biophysical and kinetic work on this process. We also saw the structure of individual domains in isolation and watched how they changed when the internet assembled. So this is the crystal structure just this domain in isolation and it is crushed together to bind the rest of the intron that has to open. So here's the closed confirmation in isolation. When it opens it is buttressed and trapped in the open state by domains two, three, four and then that opens the space between these two which then enables domain five to fit into the cleft and present itself as the active site. One of the things about this that's really interesting in this that we found between many RNAs this suggests that a lot of complex RNAs have a first counts, first folds strategy. So the first thing that gets transcribed actually folds first and forms a scaffold that promotes the faithful assembly of downstream units because RNA can missfold and make a mess really easily because it's secondary structural units are kind of metastable. You can form many different possible stable RNA secondary structures. So hardwired into the transcriptional program of an RNA is often information about this folding pathway and this really helps prevent this folding and group two introns rarely missfold. Okay, so we had learned a lot from this group two intron that we had derived from the most primitive form of group two intron called class C and it's the most ancient form. Since that time, new structures have been reporting of even bigger ones. So a more modern and larger, more developed class is class 2B and a member of this class was recently solved by my former postdoc in his own lab, Nator. And you can see it's a much larger challenge. The core architecture is identical to what we see in the 2C. And then, and this is a thing that will come up a lot in the talk, departing from crystallography more recently, Han Wei-Wang's lab in Beijing solved the structure of a 2A1 in complex with his mattress protein and that shows you the third class, the other very advanced class of group two intron. So now we know what all three of them look like. Only in this case do we know how the structure changes through the different stages of splicing and I'll talk to you about that a little more. But basically, the field has moved down real quickly so that we can sort of visualize the entire family of splicing machines and how it's diversified in time. Okay, so let's talk about determining the mechanism of chemical catalysis by this intron. Joe Cacherilli, as I said, did experiments about 10 years ago that suggested that this splices through a two-metal ion mechanism. And my very first structures on this actually show that there were two metals that were four and each one's a part. But we still needed to take a very close look at this through our crystallographic investigations. So how is the active site actually organized? Let's take a step back and think about that. So you can tell just from phylogenetic conservation that the most conserved parts of the intron are the main five and the small junction between the domains two and three were actually equally conserved and this puzzled me for a long time. What is going on with those nucleotides that they would be just as conserved as the most important parts of the intron? The answer came with the fact that they actually form one structural unit. These junction regions form a major group of triple helix with the base of domain five and then a loop nearby forms the top of that triple helix. Until this time, people didn't really think that many molecules could make triple helices in the major group because the major group is not that accessible to a transplant. It can occur and can do it over a short span. Anyway, this platform then enables another part of domain five adjacent to it to adopt a backbone configuration in which the sugar phosphate backbone curls very tightly upon itself. This is the tightest cape that's been seen in any RNA structure. And when you cram the phosphates together this closely by pulling the bases away, you create this intense electrostatic pocket that's reaching a very strong and electronegative density. It's very focused and it binds two metal ions and they're exactly four n-streams apart, okay? And we observe these in the native map just to go on density and then we confirm them through anomalous scattering of the terbium and other metals that mimic magnesium at this position. So these suggest that the intron performs catalysis as Joe suggested through that type of mechanism but we needed to be able to see it with an RNA substrate in the active site. Here you have a bi-prime splice site that crystallized in and attached to the intron and you can see this is the sysile linkage. It's perched right above the two metals and exactly the orientation that it should be for this kind of mechanism. So this was a while ago and there were a couple of things that bothered me about this data and that was the fact that in the neighborhood, like right around here and here, there was additional electron density and we didn't have the resolution and we didn't really have the data that explained what they were. So we kind of went back to work, we built new constructs that would crystallize and diffract a higher resolution and we got down to 2.7, 2.8 n-stream resolution and we began to see density that was consistent based on bond distances with monobilane potassium. Potassium has been seen in RNA structures frequently. It's usually completely dehydrated. In other words, RNA atoms often interact directly with potassium and these bond distances were appropriate for that. This was an interesting structure that we got because for two double-ion mechanisms, enzymes, it's rare to get a structure that's empty in which the metals are still there even when the substrate is not. So this was kind of an unusual, and we were excited about that. In crystallography, in contrast with priori-EL, you can actually nail down the physician's metal ions by exchanging these weak scatterers with something that's a big heavy metal ion that will scatter electrons in the gas. And for anomalous scattering, we give you a strong signal. There is an ion that's exactly the same size as potassium and that's valium. It's also super toxic, so you've got to be careful. So we thought, well, does this thing splice in valium? Another good one is rubidium. So I figured when I put together this top, there was too much structure. There was not enough actual reactions, so I want to show you the splicing gel. I'm going to show you first here the splicing of an OI intron in the presence of its typical ions, which are potassium and magnesium. This is the precursor. It hasn't spliced yet. This is basically the intron product and the largest exon product. Now, group two introns of many enzymes, many, do not function in sodium. Sodium poisons a lot of different enzymes, not just iron and enzymes. And that's because we're potassium and magnesium estes. Most of our cells are really the most sort of highest concentration of ions or involved these two ions. So then I said, well, what happens when we replace this potassium with valium? So we saw that it actually loves valium. Not many things actually function better in valium. This guy really liked valium. He liked rubidium. And then we solved a whole bunch of new structures. We solved 14, up to about 20 additional structures of the same construct in these different ions and different ion combinations. So I just cut to this one. The valium and magnesium case, when you then look at the difference map for melanoma scattering in that case, you see mass of the melanoma scattering in the valium case, particularly at this site. And also pretty strong at this site. You also see it in rubidium. And this understands that in the cesium as well. So what this did is it enabled us to unambiguously assign those positions of electron density right in the act of, say, as being monovial capitals. That was amazing because it meant that this wasn't just a simple two-metalline mechanism. It meant that group two introns capitalized splicing using a heteronuclear metal ion cluster, which like kind of more advanced protein enzymes. And I'll show you by that here. A metal ion cluster is a group of metals that share ligands and help to coordinate as the binding site of the enzyme that they're in. And K1 is part of the cluster. It's shared with M1 and M2. And in fact, what we see in structures that I'm not, I don't have time to show you, when you blow out this site, M1 and M2 can no longer bind. They're gone. You have empty introns. And so what K1 does is it helps to organize the ligands such that they will effectively bind at M2. And M1 is additionally stabilized by these. And in one of our structures, we actually can see water undergoing inline attack on a 5-prime splice site. And what you can see then, when you leave that in another structure, you can see that then the cleaved product, which is a 5-prime phosphate, is then organized by K2. So in the first stage of the first step of splicing, you get inline attack and the involvement of K1. And then K2 plays a role in managing the product after that first step and organizing them to get ready for the second step. So this was exciting because it meant that all of these metals in this vicinity have a role to play in chemistry. So up to this point, what did we learn? It's taught us that monovalence can play a major role in RNA chemistry. Potassium is really, really important in RNA structure in catalysis. K1 and K2 each play a distinct role. Rooftos are not simple two-metallion enzymes. They contain a heteronuclear cluster. And what I'm not showing you is that you often see in protein enzymes that have two-metallion mechanisms, you'll see lysine side chains in the same position as my potassiums are within the active site. So charges are placed at those same positions that do the same thing. Okay, so what were the implications of any of this for evolution? Typically, our crystal structure told us that we had a set of tertiary interactions that made a triple helix between part of the two-intron into being five. You see that in multiple two-introns and then we did a phylogenetic analysis of the U6 spliceosomal RNA. And we predicted and saw a similar co-variation between part of the catalapse, which is a concerted part of U6, and the base of the stem that it makes, U2. And then there's a similar bulge in U6 that binds megalions. And part of that bulge we predicted to make a center of a triple interaction. So we made a prediction that the U6 spliceosomal RNA is going to be in the substructure that's a lot like domain 5. And using this basic idea as sort of a roadmap, Joe Bichirilli and John Staley's lab made a metal ion specificity switches and mutational changes in the triple helices and basically showed that the exact same arrangement of metals and triple helical bases is present in this spliceosomal, okay? So through these two papers, they basically were able to demonstrate that the nodes and bolts of this active site are carried over into this much more advanced one. And there were already hints of this. And early structural work by Sam and by Dave Brown, others who were looking at the U6 structure and not just in isolation, but in conflicts with the variety of proteins that it's going to have a very domain 5 architecture. And this suggested again, that there was a parity between those two systems. And then more recently as we'll see, these spliceosomal architecture has been elucidated by cryo electron microscopy. And in those structures you see the triple helix we elucidated, you see the locations of the metals. So this suggested that in terms of evolutionary relatedness, group two introns are related to spliceosomes through their RNA components. The heart of the active site is very similar in both of them. But what about the fact that group two introns use mattress protein and spliceosomes are full of proteins? Is there a relationship between these two? So group two introns have protein partners. And as I said, they're encoded by domain four and they come back and bind to their parent intron. And we already knew that mattresses were sort of like a sophisticated form of a reverse transcriptase enzyme. There's a portion of the mattress that is basically a reverse transcriptase domain with fingers and palm. And then an X domain that functions basically as a thumb. And what I'm showing you here is just the HIV or T-dome. And so based on analysis of sequence, it was pretty clear that mattresses had a voyage on some kind of RT. This part is important for binding, this part is splicing. So that you knew the thumb played a role in facilitating the splicing and retro transposition that you didn't know would apply. The big question was, are there parts of the spliceosome that look like these mattress proteins? And so in this favor, these investigators actually were able to build an alignment of mattress proteins that wind up nicely with a protein called PRPA that is always right at the center of action when spliceosomes undergo catalysis and chemistry catalysis. And you can see that there's really, really good conservation between these two, suggesting that part of PRPA is a sort of reverse transcriptase-like domain. Okay, so up to this point, nobody may be able to solve the structure of a root two entrant mattress. Or any of the related reverse transcriptase. Because they're also related to reverse transcriptases that are important in L1 retro transposites. So line elements, other things, which make up 10% of our genome. So this is an important family of reverse transcriptases and they were uncharacterized. So because we failed to do the classical ones, we actually used bioinformatics with a bunch of different criteria, such as these, to identify ultra-stable mattress proteins from the database. And we came across two that are actually found in human gut bacteria. Not in any kind of cancer-driven bio or anything else. And these guys crystallized to beautiful resolution. And I actually have to show it to you, because as you may have noticed, we've mostly been doing RNA crystallography and resolution. And we're really happy when it's in the high twos. So I haven't seen beautiful data much in my life. So when these guys crystallized to 1.2 angstroms resolution, we were really thrilled to actually have nice data to work with. So I have to actually show you that data. Anyway, so when you solve the structure, sure enough, these guys are beautiful reverse transcriptase-like domains. They have a finger on domain. And we also were able to thread up the one element and other non-LTR vector transpose on reverse transcriptases. So these represent a first-in-class type of structure. They all this crystallize as dimers, which in our hands, we find to be functional. So this dimer interface, we believe plays a role in splicing and vector transposition. We can talk about that later. These are fully active reverse transcriptases. The active site is quite canonical with other reverse transcriptases. And I'm just showing you this to be braggy. Like, here is an aromatic side-chain for a whole minute. So that's your tool, isn't it? But anyway, here's the catalytic medallion in RT. This is a very processive, very active reverse transcriptase. And it's even active. Not so processive, but it's even. Here's the full length, and my gel is a little curved. The construct that lasts the thumb is even a reactive reverse transcriptase showing that the thumb is a processive new vector. OK, so why was this result important? This result was important because when we then looked at homology, structural homology, using a dolly search between our RT and anything else in the database that was an RT, the closest relative was the RT-like domain from PRP8 and spliceosome, which had been solved earlier. In fact, other RTs from HIV and other things were gary divergent. In fact, the only thing that comes close to these two guys are RNA-dependent RNA polymerases from polygomerases. So clearly, group 20-term naturalases and the sort of protein heart of the spliceosome are basically related. And those guys are not related to conventional reverse transcriptases, sometimes from natural viruses. They're related to RNA polymerases, which is kind of cool. OK, so I'm going to try to look through the rest of this so you guys don't fade away. Basically, this is just showing you the relatedness amongst the naturase and the RT-RPs and PRP8 by a dolly search. And so not only are the RNA components of group 20-term related to spliceosomes, but the protein components are related as well. And how they fit together to catalyze splicing in a cell, that's the next question. And we now know that, actually. And we know that from the revolution in private electron microscopy, because in that case, we can visualize an entire spliceosome. And this is the structure that was reported in nature from Kiyoshi Nagai's lab, mostly because he made a beautiful primal session, which you need to know in the next slide. And this is the group 2A in Tron, the Sulfur cryoen from Hanley-Wong's lab. And you can see that the RNA active site here is in red. And it interacts very similarly with the RT domain of both. And it's hard to see this, because everything is so big here. But what I can tell you is the most important part of the protein's interaction in both cases is that this thumb domain is interacting with the position where the 5-prime metson is recognized. So the 5-prime splice site herons are stabilized by the thumb domain in the group 2 in Tron, RTs. You can see that in here, and I'm kind of pulling it out here. So here's domain 5, here's the 5-prime metson, and the elements that recognize that. So that is primarily how maturase stimulates splice in the retro transposition. Here again, I'm showing you similarities or the different RT domains and RTs that can crystallize or salt the cryoen in only systems. And basically what I want to show you here is that even in the splice's domain, this is the thumb domain from that RT-like protein. And you can see that it's interacting with the 5-prime splice site herons that are made for you. So this is the similar type of interaction that we see between the 2-prime 5-prime splice sites and its recognition site with the 2-prime 1. So in both cases, the nuts and bolts of proteins and the RNAs even interact with each other using similar strategies. And this is just more about how the active sites are similar comparing group 2 domain 5 with 5-prime splice site with the splice's own, they're very, very similar. They both have the triple helix. They both have the diagonal metal ions, so we should talk about that. That's hard to nail down by currently yet. And again, here it's the 5-prime 1. So it's the same location. So what this boils down to is that all these different pre-MRNA splicing machines are basically undergoing catalysis using a related set of machinery. So these are related ribosomes, as it were. And all these splicing machines are metallo-inside, and they use a very similar strategy with the 2 diagonal and metal ions that are important for cleavage and elongation reactions. This all means that RNA splicing is very ancient and it originates from invasion by transposon. Remember, group 2 introns are mobile genetic elements that just came from sort of parasitic element that are initially inhabitable bacteria and then was brought into imperivance during sort of early stages of the imperiative evolution. So this RNA splicing can evolve in a unit molecular process as in the 2-introns to something that proceeds with multiple turnover. And then the splice's own is nice. It's able to actually act on multiple splice sites. And so it's acting like a true enzyme, which gives it the capability to break that one gene, one protein barrier. And RNA's splicing reactions are stimulated by ancient family proteins. And these RT-like proteins didn't originate from other RTs. They originated from viral RNA from embraces. And so that means that in both cases, eukaryotic RNA-pricing processing machine came about through the taming of parasitic genetic elements in one case of transposon and another case of RNA virus. And so from that, we got our RNA processing machine. It's a pretty good deal. And so I'll finish with that. And I will thank the grad student who did almost all the new work that I talked about today, Chen Zhao, who's now a student in my lab. Although Mark Lamarcia, this picture appeared earlier, and he's a group leader now at the EMEL, he did the work on the metal ion interactions during the scattering. We're also indebted to Matt Tuor, who was the pioneer in uterine drug crystallography, studying in my lab over at the ROHA, to treat St. Morifu, who's now a PI at Drexel, and Joe Lieberman, who were really indebted to the teams at NEPAP, the APS, HMI, NIH, and then if you want to see more about what we do, just go to my website, which we try to keep current. And then I also just want to thank the whole lab. This is on our recent kayaking trip. Go right off of the 1.9% of our own water lab. And thanks to you for your attention, you know. Do the match races ever decrease the requirements for the accessory on the test, sort of helping the RNA folder? Do you think there's always need for those accessory potassiums that are so important? You did activity with the match rays. We're not really seeing that much change in the ionic requirements in uterine. So that's probably alleviated by other proteins in the cell. But what we have seen that's pretty interesting, in which I didn't really go into it all, is that in the absence of match rays, group two latrons in a test tube tend to splice using a hydrolysis reaction. They use water in the first step of splicing instead of the branching reaction. When you throw in the match rays, it's entirely two-branching and polarity formation. And it's like 100 times faster. So, yeah, we haven't published that yet, but it's really exciting. I mean, it's a vast stimulation. But still, the chemistry is done by the RNA, but really I think the match rays is contributing to just architectural organization, the brain, where the atoms together work better. Why would the match rays pick out stable match rays that you thought would be more likely to crystallize in the absence of structure? Yeah, you know, there are just lots of programs that you can use to grind your sequences through, to look for short regions in which the protein is likely to just be natively unfolded. Kind of the isoelectric point has certain contributions. There are just many criteria that are indicative of a more stable protein that you can just apply iteratively to sequences. And just like the final show, Chen basically just went out and down. She had a few winners, like three winners. These were the ones that we went after first. But what's exciting to us, that I didn't talk about, is that this is not a thermophilic protein, but it is really stable. And it's a fantastic protein. In fact, we're going to actually distribute it so you can do end-to-end RNA sequencing. Groups of entrons, RTs are nice because they're used to plowing through structured RNA. So this guy that I described today will copy the entire hepatitis C virus genome, which is 10,000 bases, very quickly end-to-end with very few awards. So they're useful enzymes. And so we're actually developing them for study, especially alternative supplies. The potassium is, for example, reminded me of Sue Chen's work, where she was able to use different salt conditions to push the reaction forward for that kind of thing. So I was then wondering two things. So one, or two, in terms of the different salt conditions, change the forward versus reverse reaction. And if you think there may be potassium acytosis, spliceosome, or could those be occupied by protein? Yeah. Okay, so this is where I should interject. Well, I'm gonna come back to the point about Sue Chen and the riskability, but I should interject something about this, spliceosome structure. You can't do anomalous scattering or related experiments yet doing cryo-electron microscopy. So it's very hard to unambiguously establish the sites of metal that's in a cryo-EM structure. So if you see a cryo-EM structure to see all those metals, you'd be very, very afraid. And, actually, we'd like to change that. There's probably some ways to plan that. So we don't know really that well. The other important thing about cryo-EM structures is that these are done on native RNAs that are isolated from an organism. The RNAs have not been transcribed in a test of them. We fold it like the ones we've done with crystallography. Native RNAs have lots of modifications on them. So people are modeling metals where there may be an RNA modification. So we still really have trouble interpreting the existing maps of the spliceosome to know where the metals are. But based on not just RNAs age, BAMH1, lots of other very well-studied protein enzymes, I think they'll probably be amino acid side-chains, positive charge side-chains that are, like, playing a role in the K-1 case. And you're right. I mean, Su Chen's results were fantastic because they basically suggested to us that the spliceosome has some mechanism for control by these auxiliary metal sites. I haven't seen a... I haven't seen... So it's hard to drive splicing in reverse unless you have a large amount of product to drive it in that reaction. Also, remember, as soon as you splice, let's imagine you reverse splice into a product, you're not going to splice out because the polymerase is gonna trap it by copying it. So there are lots of, in the group two system, lots of systems for trapping you in the forward direction or the reverse reaction, and the same is true in the splice zone. Because, as you know, the splice's own reactions can go in the reverse tip. So, and yet, reversibility, we think maybe a mechanism for proofreading in both systems up to the point where we trap it. So it may be useful.