 Good morning. So welcome to today's lecture. So we were discussing about basics of solid state NMR and how we can apply this in structural biology. If you remember in the previous class we have discussed the differences between solid and liquid state NMR. One of the major difference that we have that in solution state most of the polarization transfer happens through J coupling. Because of the fast tumbling of the molecules these interactions that are present are averaged out and that is why we have a prominent mode of magnetization transfer is via J coupling. However in solid because of lack of motion we have a dipolar coupling and quadcopolar coupling and that leads to anisotropic interactions. However in liquid state or solution state these anisotropic interactions are averaged out we have only isotropic interaction. Then we looked at that if we want to achieve high resolution we have to adopt one technique called magic angle spinning which mimics the tumbling like situation what we have in solution and due to this magic angle spinning we can achieve sharper line. One other important differences that we looked at that inherently we detect on proton in liquid state. However because of the broad lines of proton we detect on carbon. Nowadays with because of fast spinning we can also detect on proton or when we spin dilute it with deuterium then we can also detect on proton but at moderate speed the mode of detection is 13C. However mode of detection in liquid state is proton and because of low gamma the sensitivity of 13C is low and that is why we have low sensitivity. So 13C detection and broad line are kind of signature of solid state NMR. However in liquid state we have a proton detection and that is how we have a high sensitivity. So just to summarize what we discussed the cross polarization and magic angle spinning these are the two basic building block of solid state NMR. Then we discussed about decoupling that basically enhances the resolution and to like a then we use recoupling to reintroduce the lost interaction that we had because of anisotropy. So we are then we use 2D correlation spectroscopy how we can employ this 2D correlation spectroscopy for resonance assignment. We briefly looked at the carbon-carbon correlation 2D spectrum and carbon nitrogen 2D experiment and today we are going to now discuss in more detail how we can use carbon-carbon correlation, carbon-nitrogen correlation for resonance assignment. Just to remember we at the moment we are talking about spinning at moderate speed not very high speed more than 60 kHz. So we are talking about 10 kHz, 15 kHz, 20 kHz where dominantly we are detecting on carbons and therefore we are trying to look at the carbon-carbon carbon nitrogen correlation spectrum. So we introduced to you the 2D carbon-carbon correlation which is known as a proton driven spin diffusion PDSD or DAAR which is dipolar assisted resistance recoupling sequences. So what we are doing we are transferring the proton magnetization to carbons relative respective carbon and we are trying to establish the carbon-carbon correlation that is what these 2 experiments does it. So just to walk through you to the PDSD experiment that we have here. So we start with a 90 degree magnetization which we see here. Now 90 degree magnetization first polarized spins into XY plane then using this cross polarization condition we transfer the magnetization on carbon. Now my magnetization at this stage is on carbon and for doing this cross polarization just to remind you again we are we are just getting this Hartmann hand matching condition in the rotating frame so that we can transfer the magnetization from proton to carbon. So now at this stage my magnetization is on carbon. So then we are introducing this 2D formalism where we are incrementing it to achieve another indirect dimension and that is how you see T1 here. During that time we are decoupling the protons. So TPPM is 2 pulse phase modulation pulse sequence is one of the decoupling sequence that we had discussed earlier. One of the decoupling sequence that decouples the proton carbon coupling. So then after that we now encoded indirectly into carbon dimension. Here during this period we are mixing the carbon-carbon magnetization it is similar like a nosy. We are mixing it in the in the z direction. So here is a 90 degree pulse and here again a 90 degree pulse. So we are just like a whatever we have seen 3 pulse experiment in liquid state you have 1 pulse, 2 pulse and 3 pulse. This is similar like 3 pulse experiment but here we are using dipolar coupling for mixing. Actually nosy also we use weak dipolar coupling. So here we are mixing the magnetization and because of that the magnetization can transfer say from C alpha to CO, C alpha to C beta, C beta to C gamma, C alpha to C gamma depending upon how long we mix it. Now mixing without any additional irradiation if it is done that is called PDSD proton driven spin diffusion. Proton driven because it is driven by protons. Protons are contributing to the magnetization and spin diffusion because spin is diffusing transferring magnetization to other spins like a C alpha is transferring to CO, C alpha is transferring to C beta and C gamma. So that is a PDSD. However many times this transfer is not that effective. So what we do? We apply additional pulse on proton which is of order of either magnetic like spinning speed half of the spinning speed that assist the recoupling. The transfer is assisted that is how it is called dipolar assisted resonance recoupling. So it recouples this facilitates recoupling and the transfer of magnetization from one spins it becomes better C alpha to CO, C alpha to C beta, C beta to C gamma. So using this simple carbon-carbon correlation spectrum we are now establishing the spectrum. This is kind of the parameters that are used. So here we have two axis say proton axis we are starting from here because proton has a high gamma so it has high sensitivity. So we excite protons using 90 degree pulse what is the typical power like if we are using 2.45 microsecond of pulse that corresponds to 100 kilohertz. So what we are doing in experiment little more experimental details we are exciting protons with a 90 degree pulse. The duration of this pulse is 2.5 microsecond that means the power that we are applying on proton is 1 divided by 2.5 into 10 multiplied with 4 and microsecond that goes here. So that will be 10 to power 6 divided by 10 and that is 10 to power 5 hertz and that is actually 100 kilohertz. So this is the power that we are applying on proton for exciting proton and why we are exciting proton because it has high gamma it is more sensitive. Next our job is to establish a cross polarization condition. Now cross polarization simultaneously we are applying a pulse on proton and on carbon with varying power. So if you look at here we are typically having 67.5 kilohertz, here we are having 50 kilohertz and duration of that cross polarization is 700 microsecond. Now if you remember here omega C we have is 50 kilohertz and omega H we are what having 67.5 kilohertz. So if we and omega R suppose we are keeping 17.5 so that becomes omega H minus omega C equal to omega R. So that is what conditions we are achieving here. Hartman hand matching condition duration of this cross polarization is 700 microsecond. Now our magnetization is on carbon during this cross polarization. This is called ramped CP because of R H in homogeneity we ramp it say 70 to 100 or 80 to 100 this is called ramp CP. So we are ramping we are changing the power typically power applied on proton is here and power applied on carbon that should have matching this condition omega H minus omega C equal to omega R. Then we have a Hartman hand matching condition in rotating condition. We are back on carbon we are doing the T1 encoding the frequency here on carbon while applying a decoupling. So decoupling power is 90 kilohertz this is called high power decoupling the TPPM is one of the pulse sequence that we discussed in the last slide. So applying this 90 kilohertz we are decoupling protons while encoding on carbon and then we are applying here again a pulse of 90 on carbon of 50 kilohertz which duration is about 5 microsecond. During this time we are mixing the carbon-carbon polarization magnetization of about 20 millisecond. So this can be done without this pulse or it can be done with this pulse. If you are doing without this pulse that is PDSD. If you are doing this pulse that is called DAR. So the power we are applying is omega R 17.5 kilohertz and that extra power that we are applying on proton that facilitates the mixing it enhances the mixing capacity and then finally we apply a 90 degree pulse on carbon of 50 kilohertz. We decouple the protons here using high power decoupling and we acquire on carbon. So this is the typically we are establishing carbon-carbon correlation using one of these two pulse sequence either PDSD or DAR. Wonderful. So what we get? Here we are getting now carbon-carbon correlation spectrum. It is similar like an OG spectrum. Here you see 13C carbon frequency. Here 13C carbon frequency. Here we have a diagonal peak and these are off diagonal peak that shows correlation. So here if you remember your chemical shifts here are threonine C alpha C beta. So this is threonine C beta. Here we have a threonine C alpha and then if you go around this axis we have a threonine C gamma. Here are the reason for serine and we can have here alanine. So sorry isolucine you can see isolucine gamma delta gamma 2 beta alpha. So isolucine you can establish all the way like here say if you go to diagonal peak here is alpha of isolucine then beta of of isolucine here then gamma 2 gamma 1 and delta. Looking at these chemical shifts you can identify spin systems and if you start mixing more and more the neighboring residue will start contributing what we have here. So you can even get it near neighbor effect. So like a C alpha of magnetization of this I residue can be also transferred to I minus 1 residue CO or C alpha depending upon how long we mix it. So it not gives only spin system specific assignment but also near neighbor assignments or even little medium range assignment depending upon how we mix it. So we need to just vary this mixing time. 20 millisecond is short distance correlation if we increase to 150 millisecond it will be neighboring residue if we increase to 500 millisecond even more some long range 800 millisecond even long range. So you can establish all these carbon-carbon correlation using DAR or PDSD that helps us in resonance assignment of this spectrum. So I am taking another example of say here K 59 K is lysine 59. So we have a C alpha here if you just follow these lines. So C alpha here in red you can get a C beta lysine has C gamma C delta C epsilon. So we can assign all those and C epsilon comes around 41 42 ppm C beta comes around 35 ppm. So if you know from BMRB values of different amino acid you can essentially analyze it. You see here epsilon of lysine is coming around 42 beta is coming here delta's, gamma's and all those are coming there and which can be assigned. So I am showing you one of the publication from Adam Lange group where they have used this BACA bacteriophiline they got a beautiful spectrum carbon-carbon correlation spectrum and you can see it is as beautiful as no G spectrum. You can use this PDSD based experiment or DAR based experiment for resonance assignment. So these are all short range correlation. You can see here is isoleucine C alpha C beta serine C alpha C beta then Q B and Q C alpha C beta. If you go here this region phenyl anionine you got this region is for alanine C alpha C beta here are isoleucine this is serine sorry threonine C alpha C gamma. So all these correlation one can easily establish from C alpha to C beta to C gamma to C delta here C beta to C gamma C delta here are delta's delta can be also shown here like you can see all the way going from top to bottom we can assign this spin system specific assignment resonance assignment or near neighbor assignments by doing couple of of months of assignment. This is beautiful spectrum and can be used for starting the assignment. But that is not enough we have to establish this was only carbon carbon spectrum. So we have to establish heteronuclear assignments for a like a carbon nitrogen experiment like we have a typically HNCO or HNCA kind of experiment in liquid state. Similarly in solid we can do this by again polarization transfer. So to explain to you how we do it I am just explaining you see ith spin system this is the I-1 spin system we start polarization from HN using dipolar coupling we transfer to like through bonded connected N15 that nitrogen of amide then we transfer to C alpha and we establish the correlation between nitrogen and C alpha this will be direct dimension here will be indirect dimension. So we are establishing a correlation NC alpha and we are detecting on carbon while nitrogen is on indirect dimension. So we have a 2D of NCA. Similarly rather going in this direction we can go in backward direction. So this will give I-1 correlation HN we are transferring to N15 going to CO. Now I-1 correlation can be established here and that will be kind of HNCO experiment that we have in liquid. So this is HNCA and this is HNCO but in solid since we are not utilizing protons we called it NCA and NCO. Here we are 2 times transferring the magnetization from HN to N15 and N15 to CO or C alpha this is called double Cp experiment XCp yaha, 2Cp yaha, first Cp here, second Cp here that is what it is called double Cp, DCp double Cp experiment. So schematic is something like this we are starting with proton and transferring to, transferring here to carbon here we are starting here. So we can get I correlation, I-th correlation we can go back and we can get I-1 correlation in NCO and the pulse sequence is something like this. So let me walk through Q to the pulse sequence. We are again exciting the protons using 90 degree pulse. Then we are doing the first Cp we are transferring the magnetization from proton to nitrogen. So first Cp directly attached protons magnetization are transferred to nitrogen, now magnetization is on nitrogen. Then we are indirectly like in direct dimension we are encoding nitrogen so this is the T1. Then we are using another Cp here for transferring but before that we are just decoupling protons as well as using 180 degree pulse we are decoupling carbon as well when the nitrogen is evolving. So then when we are at this stage we will do use the double Cp like the second Cp and we transfer the magnetization from nitrogen to proton while we decouple again proton, sorry nitrogen to carbon while decouple proton and then we detect on carbon. So this is our T2 and we are again decoupling protons. So these are the two high-powered decoupling we are using. One when T1 is being encoded when T2 is being encoded and during this transfer we are using the Cw decoupling. So that is why we have a one dimension nitrogen 15 another dimension carbon 30 and depending upon how we want to transfer the magnetization the same pulse sequence where we shift our the carrier frequency the NCA correlation can be established or NCO correlation can be established. Same pulse sequence just you have to shift the frequency. If you are shifting on CO we can establish the CO correlation I minus 1 NCO correlation whenever shifting to 55 ppm then we can establish the CA correlation so that would be NCA correlation and when we are shifting to say 175 ppm it would be NCO correlation. So that is how we do it and when we do we get a beautiful spectrum this is from Chris Zeronic group they have done on some fibril protein you can see now beautiful again NCO correlation you are getting this dimension is N15 this dimension is carbon 13 and here for each amino acid I minus 1 correlation we are getting here for each amino acid ith correlation we are getting. So here is like you know this is CO of say 137 this is CA of 137 similarly we can get for valine so you can see CO of 122 valine so similarly you can establish I minus 1 correlation coming from here and ith correlation coming from here just by looking at this and using couple of those carbon-carbon correlation spectrum you can assign resonance sign all of you can identify all of these correlation beautiful but that is not enough right we just did 2d we need to establish further correlations to get a resonance specific assignment so what we can do the kind we fuse this first we perform the carbon-carbon correlation now we are performing nitrogen carbon correlation can we fuse little bit this and get a more magnetization transfer that is what these experiments do NCO CX and NCO CX so what we are doing here starting with a proton we are transferring to N15 encoding here and then then we can transfer to CO but we do not stop it here let it mix whatever we are doing in pdsd or dark so we can fuse these two sequence the 2d HNCA correlation with pdsd or dark correlation so now my magnetization does not stay at CA it can go all the way to C beta, C gamma, C delta so that will be called NCA CX when C beta, C gamma, C delta and all NCO CX analogous to like our HN this is like HNCACB this is like HNCO CACB so here we are transferring starting from proton transferring to nitrogen going to I-1 CO and then we are mixing that so we can get the I-1 C alpha, C beta, C gamma and C delta the third one can be also depending upon how we are starting so starting from say H alpha, C alpha, N then going to CO and then mixing with CX so this is like HCA, NCO, CACB something like that so I-1 all the correlation we are getting when we are starting from H alpha to C alpha to N15 going to I-1 residue CO, C alpha, C beta, C gamma, C beta so these are these can be 2d version as well as in 3d version depending upon how many axis or frequency we are labeling it now using all these series of these experiments we can almost achieve a liquid like correlation spectrum which can be used in a similar manner for resonance assignment just one of the example that I am showing here so NCO, Cax and NCO, Cax so if you look at here it was NCA experiment but when we did PDSD, NCO, CX, PDSD now we are getting also correlation from C beta, C gamma and C delta take any amino acid that we want K60 here you see it is C beta is coming here C gamma and all those so here similarly one can take valine here valine you get a valine 53 or something like that C gamma, C delta and all those so glycine just one peak coming here so you can establish putting NCO, Cax, NCO, Cax even using 2d you can achieve lots of lots of resonance assignment because 2d can be done in few hours 3d will take days so you can collect a good 2d and that should be enough for resonance assignments of few of these peaks. So here I show an example how you can use your DAR or PDSD with NCO, Cax and NCO, Cax and establishing the correlation so you can start from say Thionine, C alpha, C beta then you can go to C gamma here you come and find it is a C beta then here from here you can come C alpha here you can find the NCO alpha we can go here and find it out CO of my I-1 residue so this helps us facilitates us in working. You can take another example like here in this region we can get an alanine so alanine C alpha is here C beta is here now we can come all the way we get N15 of this we can get I-1 of this and similarly using NCO, Cax, NCO, Cax we can assign each of these resonances that are present this is a few weeks or maybe a month job depending upon how much experience you have this can be used for resonance assignment. Now people did not stop it there they developed a series of sequence so one of the sequence that I am showing from Tata-Gopinath and Vagilia that developed the carbon 13 edited C-C experiment and 15 edited N-C experiment on N-C-S-C-X experiment like N-C-S-C-X, N-C-O-C-A-X, N-C-S-C-A-X, N-C-A, N-C-O-N-C-S-C-X, N-C-O-C-X, N-C-S-C-A-B and many of these where sometimes it is also labeling the protons. So this is a gallery of experiment that they have developed and these can be essentially used for doing resonance assignment. Tata-Gopinath use multiple equations so in actually in the same sequence he can detect various of various experiment like C-S-C-X, N-C-S-C-A-B, N-C-S-C-X, N-C-O-C-X. So in same experiment at the different time point you can detect it various experiments so you save lots of time because these are low sensitive experiment. This is a beautiful exposition of Tata-Gopinath and Vagilia tried to attempt to cut short the times like cut drastically the acquisition time and facilitating resonance assignments in a shorter possible time. Now we have to if we record all these series of experiment you can take a strips right so you can take a strips and you can sequentially work C alpha to C beta, C delta to C gamma using various experiment like N-C-S-C-X, N-C-O-C-X, C-A-N-C-O, N-C-S-O all these are you can see in different colors these are there and similarly like liquid state you can use this strip for resonance assignment. So 128 we can work to 127 we can work to like using these assignments 126, 125, 124, 123. So if we assign all those now we have the value of C alpha, C beta, C-O then what is the obvious next step then we can get these assignments and we know that these chemical shifts are quite deterministic of the secondary structural information. So we know from our chemical shift that C alpha has only glycine right. So however these spin system like alanine, therene, thionine, serine they have C alpha, C beta and they have a characteristic C alpha, C beta so it helps us in resonance assignment. Glycine has C alpha only at 45, alanine has C alpha around 52 and C beta around 22 just two spin system, thionine C beta is lower than C alpha, serine again C beta is lower than C alpha. So using all those signatures we can start the assignment these are start point or check point. Similarly C alpha, C beta and C gamma gives most of other amino acids, histidine, tryptophan, tyrosine, phyline, alanine are difficult to identify because they have a broad line but then we can combine their aromatic chemical shift to find it out. And then looking at these distribution of different chemical shift for different amino acid that we have seen it becomes quite easy for assigning using the spectrum of 2D or that we have combined here like a 2D you can start from like that is what I always showing you started from thionine it has a distinct chemical shift serine has a distinct alanine has a distinct glycine again comes somewhere here. So you can use these distinct chemical shift for resonance assignments and then you once you have the resonance assignment then subtract the sequence corrected random coil chemical shift of say C alpha, C beta plot along the sequence. So here what I am showing you again from Madam Lange group the delta C alpha minus delta C beta so these are C alpha and C beta subtracted the random coil chemical shift you plot along the sequence and then if these values are negative that you know that these are beta sheet. So this protein that I am showing you is in reach of beta sheet you can see using the secondary chemical shift you can plot along the sequence and you can find it out what is the secondary structural topology similarly like liquid state. So we got a secondary structural topology what next? Next is now we establish the secondary structural topology can we get inter sheets or inter strands the distances if we fix these distances then we are all set for a structure determination of these long elongated fibroler structure that we have. So for that I said PDSD is also good enough because that gives you long range correlation some of these distances we have to measure using PDSD. But in the next class I am going to show you how what else we can use for measuring the long range distances but PDSD is good enough for giving you long range distance you can see 107 to V97 these kind of distances you can even get it from PDSD here 100 to 117 the long range distances can emerge from carbon-carbon correlation and that fix us measuring or getting the topology called arrangement of these beta strands how they will be arranged that is how we can generate a structural model of any fibroler. So with this I am going to stop it here today and in the next class we will be taking we will be taking the idea of how we are going to use these structural information to getting the due de novo structure of these difficult protein system whether they are amyloid fibres or membrane protein in their native state or the native setting. So I hope to see you in the next class. Thank you very much.