 So, welcome to today's class. In the last class, we discussed the basics of solid state NMR spectroscopy and how we are going to use that in structural biology. So I introduce you the difference between solid state NMR and liquid state NMR spectroscopy and what are the protein molecules that are applicable for solid state NMR where we can apply solid state NMR for looking at the structural and dynamic aspects of biological macromolecules. I also discussed about inherent problem with solid state NMR and what can be toolkit for getting some of these problems, overcoming some of those problems. So let us repeat little bit and then we move forward. So in the previous class, I discussed that we want to mimic the inherent averaging process that is in solution state NMR to obtain high resolution isotropic information. So inherent averaging process that is present in solid state NMR. If you do that, if we mimic that solution state like condition, then we can get the isotropic information and this to do that what we have to do? We have to enhance the resolution and sensitivity. So how we are going to enhance the resolution and sensitivity by removing the anisotropic part that were present in solid and retain only isotropic part. If you remove an isotropic part, retain only isotropic part by something called decoupling or averaging of interaction, then we can get a sharper line. But by doing this, we are like a getting rid of important structural parameters. So we have to introduce those, get back those in isotropic part for elucidation of geometric parameter like a dipolar coupling or chemical shift in isotropy. So that is called recoupling or reintroducing these information. So first we decouple to get sharper lines, then we recouple to get important structural geometric parameter. With doing this, we can achieve best out of both world, the sharper line which is generally seen in the solution state NMR and all the structural parameter that are present inherently present in the solids. So if you do that, we can achieve the structural aspects or dynamics, emotional aspects of protein molecules in solid state NMR. So how to do it, actually it all started when John Kendrew in 1958 proposed this idea of a spinning sample at an angle which is called magic angle. So you know this is our main magnetic field B0 and in solution state we know that our sample tube was something like this. So all the spins were aligned like this. So our spins were placed, samples were placed along the magnetic field. Now John Kendrew proposed since dipolar coupling is culprit, so can we do something? Can we orient our sample at an angle which is called a magic angle, this theta m in the magnetic field and then we spin them faster and faster and faster and faster. Because of this faster spinning, the interactions that we talked will be averaged out. This theta m is a magic angle. Why this is magic angle? Because you know 3 cos square theta minus 1 is that dipolar coupling term and if you want to make it average, so we have to put it 0. So now you can calculate what will be our theta, that will be 54.7 degree. So if we orient our sample at that angle which is called magic angle, we are putting our sample in this rotor. This is the spins that are in the rotor and we have to spin with air faster and faster and because of that actually this anisotropic interactions that is there will be averaged out. So what are the samples where we are putting samples? All sorts of samples whether it is membrane protein or fibril protein or aggregated protein any of these we pack in this rotor. That rotor is placed at an angle called magic angle in the magnet and then we are spinning and you see what happens because of this spinning here. So if we spin it, if we just let me just remove this, just one minute. So because of this spinning at magic angle. So let us say in a static case we have seen right the peaks were really really broad and I am taking the simplest sample glycine where there are only two carbon one with CH2 that is alpha carbon and one carbonyl carbon CO. So for alpha carbon we are getting one peak here really broad peak here and then for CO we are getting a really broad peak here that is for a static case if we are not spinning. It is a glycine the simplest biological molecule that you can think of amino acid. When we start spinning, so we are spinning speed is here say 0.85 kilohertz that is a kilohertz means like this many rotation per second then lines starts becoming really like little sharper and you can see now these whatever we have discussed orientation dependence chemical shift is start appearing here. If we start little faster like a 3 kilohertz we see now like are slowly getting sharper. If we go to 5.5 kilohertz two lines are now really really become really have become sharp and if you go to 12 kilohertz we see only two peaks beautiful two peaks one coming here at the C alpha one coming here at the CO. So glycine is spinning at 12 kilohertz is giving us really sharp peak so we have to spin it. You remember this 12 kilohertz means 12,000 rotation per second. So this is quite fast spinning if you compare with your ultra centrifuge that is one like RPM that is rotation per minute here we are talking 12,000 rotation per second. So this is damn fast you have to spin that done damn fast. So this material that is there that is made has to be really robust and the mechanical aspects also has to be very robust. So these are a spin with air and that is given from outside and here are the fins and it has to spin very stable in a narrow passage. So this is made up of zirconium oxide it is a really robust material because we are doing experiment at various temperatures so it should be temperature insensitive and it can spin really really fast. So this is one of the moderate spin that we can think of 12 kilohertz it can now it can go up to 110 kilohertz and that is a special arrangement that you need for spinning. But because of this spinning now we are going to get really sharp lines. Yeah so as we discussed we have to spin it spin it very faster and this spinning is called magic angle spinning. We are spinning our rotors at an angle which is called magic angle and this actually averaging out our anisotropic interactions. So now we have a different kind of rotor because for depending upon samples we have to spin it at different speed and we have all the way like this is say 7 mm then 4 mm rotor. So this is 7 mm means 7 millimeter is the outer diameter of this rotor then 4 mm, 3.2 mm, 2.5 mm, 1.3 mm or even like you can see now a days we have a 0.8 mm rotor that is really really fast spinning rotor. So here is a 3.2 mm and you need a nano gram of sample for doing solid state remand just with a coin this are compared. So if you reduce your outer diameter that means we can spin faster and faster. So outer diameter of magic angle spinning rotors determine the maximum rotation frequency like if we are having 6 mm rotor that means we can spin maximum to 8 kHz. If we have 4 millimeter rotor we can spin up to 15 kHz. If we have 2.5 mm rotor we can spin up to 30 kHz. If we are going say 1.9 kHz, 1.9 millimeter of rotor we can spin up to 40 kHz, 42 kHz and if we are spinning up to like 1.3 we can go all the way up to 65 to 70 kHz. And now the fastest rotor, the fastest available rotor is 0.8 mm that can go 100 or 110 kHz. So now this different size of rotors are essential for spinning faster and faster. Now since we are miniaturizing this rotor size, so that means the sample that we can pack in this is also going to be very very small. The sample volume depends upon what is the inner diameter of this rotor. What I talk to you is outer diameter of the rotor. So if inner diameter if we talk that is going to be very small because this material has to be very stable that is why it is made up of z-conium oxide. Now these are two, this is the top cap which has a fins that drives the spinning. So essential volume that like the effective volume that is there is very small for these smaller rotors. So and the mass frequency also depends upon what is kind of the size, so smaller size faster rotation, bigger size slower rotation. That also determines what kind of experiment that we are going to implement. Here we can like since we are spinning fast, so we can average out really lots of the stronger interaction and therefore we can do something called even proton detection that probably I am going to talk to you briefly. Here mostly we have to do carbon detection. 3.2 rotor can spin up to 42 kilohertz and that has a speed of 240 meter per second when rolling around along the ground and that needs only 46 hours to roll around the whole earth. That is the fastest, that is the fastest I am talking. So we have to spin these rotors really really fast to average out the isotropic interactions that we have talked about and because of this we are going to get basically the sharper lines that we have talked. So the powder pattern gives us broad line and the side band but we have if we are spinning faster then we can get a sharper line and this mass frequency is chosen so that we get out of this side band and then we can then once we get a sharper line then we can introduce some of the recoupling experiment that I have talked to you earlier. So how we do? So you remember we are doing two things. We are first decoupling the unwanted interaction and then selectively introducing the wanted interactions. So decoupling so suppose here is one spin coupled with four spins so here is a rare spins in red which we can call it carbon 13 or N15 and that is surrounded by abundant spin protons. So we have homonuclear dipolar coupling between protons and we have a heteronuclear dipolar coupling between carbon and proton. This is relatively weaker coupling and this is very like a quite strong coupling. So if we spin faster and faster we can like weaken these couplings. So if you look at the order of the dipolar coupling proton-proton since the proton-proton distance is shorter about 1.8 angstrom the coupling in terms of kilohertz is about 21 kilohertz. The carbon-proton coupling the distance is 1.1 the here coupling is about 23 and another if the distance is like increasing up to 2 you have a weak coupling like 3.8. So depending upon what is the distance between these two dipoles your coupling also varies and because of this the mass we can weaken these coupling and then we can do something called decoupling we like selectively irradiate these couplings by a series of pulses which is called decoupling pulses then we can get rid of these heteronuclear coupling. So because we are getting rid of this heteronuclear coupling we are going to get a sharper line. So that is what we do in a typical experiment in heteronuclear decoupling experiment the first thing just to take you on track first thing we did magic angle spinning we put our sample in this rotor we put that in the magnet at an angle which is called magic angle and then we are spinning faster and faster and faster depending upon what is our requirement all the way from 10 kilohertz to 100 kilohertz that average out some of those heteronuclear couplings. Then next thing we are doing is dipolar decoupling what we do in this dipolar decoupling so we always start polarization from the proton which is a eye spin and then we transfer that we can transfer that on carbon typically that I am going to talk to you in the next slide but in decoupling what we do we selectively irradiate in decoupling this is the simple thing we irradiate the proton and we detect on carbon because of this irradiation constant or constantly irradiation on proton we can we are getting rid of these the heteronuclear dipolar coupling. So in this simple experiment what we are doing we are having to spin one proton one carbon proton is eye spin carbon is a spin we are applying a 90 degree pulse detecting carbon while decoupling protons. So S spin is detected and eye spin is decoupled now this is what we get a sharper line. So because of magic angle we are getting this weakened coupling because of irradiation we are removing this these couplings so and now this because of this RF application we have removed this coupling and therefore this is kind of a isolated spin which probably gives us which is going to give us better resolution. The next important parameter that is in solid state is cross polarization this is something like Hartmann-Hahn condition that we have discussed in liquid state. So cross polarization is what you know the proton the gyromagnetic ratio is high carbon it is a one-fourth that is more sensitive carbon is less sensitive can we exploit that more sensitivity of proton for our benefit and for doing that what we do is called cross polarization we are polarizing proton and carbon simultaneously and transferring that polarization from proton to carbon to enhance the sensitivity. So how we do that let us start again with two spins eye spin and S spin first we are exciting protons using 90 degree here now proton is excited so this is like we apply X pulse so it is in Y. Now then we are simultaneously applying the RF on proton and carbon and this is ramped actually ramped because of because of it gives the stable performance at high mass. So we are ramping here and then we are matching that Hartmann-Hahn condition if you match that condition now the polarization from proton is transferred to carbon so we have enhanced the sensitivity. Now we will decouple the proton and detect on carbon so we did two things we have started with proton transfer our magnetization to carbon using this cross polarization we decouple proton and then detected on carbon. So because of this we are enhancing the sensitivity of carbon so resolution was enhanced using magic angle spinning and decoupling and cross polarization is increasing the sensitivity. So these two are basic building block for any solid state NMR experiment. The cross polarization CP and magic angle spinning that is mass so what is the cross polarization condition? So you have to have these delta omega nutation that is spinning frequency should match with the rotational frequency r or 2 omega r delta omega h minus delta omega carbon should be matching with our delta omega r in spinning condition to have this Hartmann-Hahn magic angle condition. So suppose we are spinning at say 10 kHz so difference between the frequency that is applied on proton and frequency applied on carbon so here is 10 kHz that should be either 10 or 20 or something like this and then we can establish this Hartmann-Hahn condition and we can increase the sensitivity. Therefore to conclude magic angle spinning is essential for getting the sharper line then it is supplemented by heteronuclear dipolar coupling so these two leads to resolution whereas cross polarization leads to sensitivity of the signal. So CP and mass these two are basic building block of any solid state NMR experiment. So as we saw this we have to do X nuclei detection that is carbon or nitrogen while decoupling the proton this proton decoupling is removing the heteronuclear dipolar coupling so we have to apply a high power decoupling on proton that is going to be order of 100 kHz. Now if we do that we are going to get a really sharp line so magic angle spinning remove an isotropic interactions and remaining was removed by this high power decoupling and then we are doing cross polarization to enhance the sensitivity. Now we are almost achieving the liquid state light spectrum so for this L valine in L phenylalanine if you look at here the liquid state spectrum shown in blue and solid state spectrum shown in red you are getting really really beautiful sharp lines almost comparable to liquid state. Now we achieve it we wanted to always have a sharper line is not it. So now we had a sharp line so what are the disadvantage of solid state why we cannot do solid state for everything this disadvantage is now because of lower sensitivity and we have because we are detecting on carbon so that is a lower sensitivity to compensate that sensitivity we really need larger samples so we are putting about 30 to 40 milligram in 3 or 4 millimeter or 3.2 millimeter rotor to get such signals and we have to probably record little longer to achieve the same signal to noise ratio for in the solid state. So these are some of the disadvantage but still we can detect these samples in their states what it should be and this is very much useful like a say for farmer sample. So many of the medicine that we consume comes in a tablet and if you dissolve them then there is a possibility that their property will change now we do not want to perturb anything just take that pack in the rotor record the spectrum and you see what kind of the molecular configuration and the orientation of these moieties are there in their formulated state. So solid state NMR is a great boon if you are doing that in the formulated state and still we are achieving very sharp lines so we can tell everything that is needed. So advantage we are getting by doing this CP and mass is really high resolution now we can see it almost compared to and we are getting all the peaks that are there in the liquid state NMR. So we detected on proton sorry carbon can be detect on proton right proton detection because sensitivity was less for carbon. If we replace that carbon with proton we are going to get it better sensitivity so can we think of detecting now proton as well. See proton the inherent property that because of high dipolar coupling we have a really broad signal here okay you can see the broad signal. Now if you are no mass no decoupling we are really getting broad signal you can see how much it is dispersed. Now we start spinning and still no decoupling then we are starts to see some features some resolution and depending upon how fast we are spinning it. If we are spinning it say 20 kilohertz still there is a line broad but if you go about 100 kilohertz we start getting these sharp features and we apply mass magic angle spinning and then do proton-proton decoupling then we are really going to get sharp lines. So for proton detection you need damn fast magic angle spinning and high power decoupling for proton-proton decoupling and still we can detect it still we can detect proton so we are not losing sensitivity but you remember I just showed you the rotors that is used for fast spinning it is a 1.3 millimeter or 0.9 or 0.8 millimeter. Now if rotor size is decreasing the OD I am talking about 0.8, 0.9 or 1.3 the rotor size is decreasing the effective volume that is available for putting your sample is also reducing. So you really need to put it small small amount of the sample inside the rotor if we can if we are happy with that if your problem is getting solved with that small nanogram of sample you can still spin very faster detect a well resolved spectrum using protons and you can get all the structural parameter. So that gives you little bit of perspective how we initiate our experiment using solid state NMR. Here onwards I will take little bit of transition go because 1D is not good enough for structural biology you know right. So we need to transition into 2D. So how we are going to use two dimensional NMR spectroscopy for a structural elucidation of bio-micro biological macromolecules in the next class and I hope you are getting along with me. So I would request you to go back and little bit read about the basics tools of solid state NMR and the next class we start with the two dimensional aspects of solid state NMR and how we can utilize those two dimensional spectrum for getting the structural information for biological macromolecules. Thank you very much let me stop it here today. Thank you.