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proton (H) of (mostly) every NH groups presents in the protein. One needs a 15N labeled protein for this experiment. Therefore, every peak in this spectrum, represents the fingerprint of each amino acid making up the protein. Note that not only the backbone amide groups are seen, but also side chain NH groups (from Arg, Asn, Gln, His and Trp, sometimes Lys) can also be present in this spectrum. CI2 sequential backbone assignment tutorial Thanks to several Utrecht NMR Research group lab members [file:///home/student/STRUCT_BIOL/ASSIGNMENT/html/assignment.html] Once NMR spectra have been recorded and processed, the following steps are to assign the peaks in your spectra. In this tutorial, you will assign the sequential backbone atoms of a protein, CI2 (Chymotrypsin Inhibitor type 2), using 2 3D-NMR experiments, HN(CO)CACB and HNCACB, and a 2D-NMR experiment, 15N-1H HSQC. SPARKY is a NMR spectra visualisation and analysis program that has been developed to assist in NMR structure determination of proteins, DNA and RNA. We will use this software for the assignment of the protein. When a double labeled sample is available; both 15N and 13C labeled; it is then possible to extend the dimension of recording. This is called 3D-NMR or triple resonance experiments. Spectra produced after recording these 3D-NMR experiments are used to assign the peaks present in the HSQC spectrum. Roughly speaking, two complementary 3D-NMR experiments are recorded, HNCACB (or equivalent CBCANH) which will give you the chemical shift information for each amide (HN and NH) peak in the HSQC spectrum of the residue (lets call it residue i) and the chemical shift information of the alpha (CA) and beta (CB) carbons of the residue (residue i) and of the previous residue (residue i-1). Note: To run this practical make sure that SPARKY is installed on your system, otherwise download it from the SPARKY website. A few helpfull links for this practical: PDB entry of 2CI2 Manual of Sparky Structure Determination by NMR Questions & assignments are indicated in bold and with a *. Please hand in the answers at the end of the practical. The second spectrum, HNcoCACB (or equivalent CBCAcoNH) will also give the chemical shift information for each amide (HN and NH) peak in the HSQC spectrum of the residue (residue i) but CI2 is a protein composed of 64 amino acids. The amino acid sequence is displayed below. only the chemical shift information of the alpha (CA) and beta (CB) carbons of the previous residue (residue i-1). Hence, you can use the information from this spectrum to assign which CA and CB are of the residue and of the previous residue in the HNCACB (or CBCANH). Sequence of CI2: 1-10 LEU LYS 11-20 LYS SER 21-30 LEU GLN 31-40 VAL LEU 41-50 GLU TYR 51-60 VAL ASP 61-64 PRO ARG THR GLU TRP PRO GLU LEU VAL GLY VAL GLU GLU ALA LYS LYS VAL ILE ASP LYS PRO GLU ALA GLN ILE ILE PRO VAL GLY THR ILE VAL THR MET ARG ILE ASP ARG VAL ARG LEU PHE LYS LEU ASP ASN ILE ALA GLN VAL VAL GLY In the 15N-1H HSQC spectrum, every peak connects the chemical shifts of the nitrogen (N) and the The use of 13C spectrum makes the protein assignment much easier for two reasons: Addition of the 13C dimension reduces the overlap of the peaks that often occurs in the 2D spectra. Since the chemical shift of the CA and CB are specific for each amino acid, it is thus possible, just with these 2 carbon resonances, to identify each amino acid and use them for the sequential backbone assignment. The average chemical shifts for the carbons alpha and beta for each amino acid is shown in the next picture. The values have been taken from the BioMagResBank. A nice tool that includes this picture is available: cd ~/STRUCT_BIOL/ASSIGNMENT/shift/ and type /usr/bin/wish in your terminal, then source shift.tcl, followed by hh_shift_table. Click on the button H/C to alter between proton and carbon chemical Go to the directory shifts. To open the project for this exercise go to File select assign.proj (located in the -> Project/Open project and /home/course*/STRUCT_BIOL /ASSIGNMENT/sparkydata/Projects/ directory). Three spectra should appear on your screen; an HSQC of CI2 and the 3D spectra; HN(CO)CACB and HNCACB. The HSQC spectrum has two scrollbars; so you can view the spectrum even when you are zoomed in. Use the shortcuts zi and zo for zooming in and out, or use the zoom pointer mode. Both 3D spectra have an extra scrollbar at the bottom, so you can go through the 3rd dimension (of the cube). In the HSQC spectrum, peaks correspond to all the amides of CI2. The 3D spectra are put in such a way that we see the 1H-13C plane of the cube. Pick a peak by typing F8 and clicking on a peak. When you select this peak in the HSQC and center on it - type vc (view center) - you will see the CA and CB carbons of this residue (and the one before) in the 3D spectra. All the spectra are connected, so when you will move through one of them, the other ones will also move. This is made possible in View -> More -> Synchronize. You will see that the 15N, 13C and 1H axis are connected. Specific View options of each spectrum can be accessed by the shortcut vt or View -> View options. The menu can also be accessed by right-clicking inside the spectrum. Contour levels can be adjusted by the shortcut ct or View -> More -> Contours Levels. These levels do not have to be changed anymore. First you have to peak-pick the HSQC spectrum. The pointer mode should be selected as find/add peaks. Now you can put peaks into the spectrum by hand. The program can also do automatic peak picking by dragging a box over the spectrum. Sparky will automatically recognise the peaks according to the current contour levels. When you put a peak by hand; the peak can be centered by the shortcut pc (peak center) and integrated by pi (peak integrate). The last one can be handy for overlapping peaks; Sparky will fit the lineshape with two or more peaks. * How many (backbone & side-chain!) peaks do you expect to find in your 1H-15N HSQC spectrum. Compare this to the number of peaks found by SPARKY Now the peaks have been picked; find the corresponding CA and CB values in the 3D spectra. Since the 3D experiment data that we have are really of good signal/noise ratio and well dispersed, we can do a restricted automatic peak picking. First select all the peaks in your HSQC and then open the Restricted Peak Picking panel (kr, or Extension -> Restricted Peak Pick). Select to find peaks in the HNCACB using peaks in the HSQC; the ranges are alright; then pick the peaks. * How many peaks do you expect to find in your HNCACB? Does the amount of found peaks match that number; if not try to explain the difference. What about the HN(CO)CACB? Analyzing the NMR spectra of CI2 with the software SPARKY You can make a so called 'strip plot' from the 3D experiments. This is a handy tool for the assignment. First select all the peaks in your HSQC and then open the Strip Plot panel (sp or Extension -> Strip Plot Peak Pick). Select the 3D spectra in Show -> Select Strip Spectra. Mark the HNCACB and HN(CO)CACB. Leave the axis order as HCN, click on OK and close the panel. Then make the strips by Show -> Add Selected Peak Strip. The strips are put next to each other and you are able to see which carbons belong to the residue itself and which belong to the previous one. * Use the chemical shift statistics (shift.tcl) to find some specific residues like Threonine (T), Open a new terminal and type sparky to start the assignment software. Serine (S), Alanine (A) or Glycine (G) and write down their 1HN, 15N, 13CA and 13CB frequencies. These residues can be used as starting point for your assignments. By looking at the previous carbons of the residue; you can already guess what kind of residue it will be according to the statistics; you can map this in the sequence of CI2. Select a peak in the strip plot and go to one of your 3D spectra. Type vc (view center) and you can see this strip in your actual spectra. phi and psi backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. These predictions will be indicated with Good. The flow chart of the TALOS program is shown in the figure below Now delete all strips and select one of the starting peaks in your HSQC. Make the strips of only this peak in the Strip Plot Panel. Now select the peaks from the previous residue (visible in both experiments) in the HNCACB and try to find matching peaks ( sm or Find -> Add Strips Matching Peaks). In this way it should be possible to go through the sequence and assign all the HSQC peaks. * Try to assign a stretch of about 5 residues in the protein and write down their 1HN, 15N, 13CA and 13CB frequencies. Remember that one can see the prolines only from the next residue and that the first residue can most of the time not be found. Use of backbone chemical shift information for predicting dihedral angles The following figure shows the graphical display of the TALOS output. Chemical shifts of backbone atoms in proteins are very sensitive to local conformation, and homologous amino acid sequences show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13CA, 13CB, 13 C', 1Ha and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar ~/STRUCT_BIOL/ASSIGNMENT/talos_ci2 directory. Look at the file called ci2d_shift.tab. This file contains the chemical shifts of the CA, CB, C, N and HN nuclei of each amino acid. Type in the terminal talos -help for a short description of TALOS. Then type talos.tcl -in ci2d_shift.tab. TALOS will start the predictions and will save them in the directory /pred. For a summary of the results, type vina.tcl -in ci2d_shift.tab -ref ci2.pdb -auto. That will create a summary file called pred.tab. You can then see the results of the prediction by typing rama.tcl -in ci2d_shift.tab -ref ci2.pdb. Go to the * How many residues have a helical conformation? And how many are located in a beta-strand? Open the crystal structure of CI2 in a graphical program like molmol: molmol ci2.pdb. Select all atoms Prop > Select All > Atom. Since in a crystal structure there are usually no H-atoms, we will have to calculate them: Calc > Atom > H*. In order to calculate the secondary structure elements and to show a ribbon presentation of the structure, do the following: Attr > Style > Bond > invisible and click on ribbon in the panel on the right. You can label the structure with the residue numbers by clicking on label num. To calculate the backbone angles in the crystal structure, write the following lines in the command-line: SelectAngle ':*@PHI,PSI' followed by CalcAngle. * Compare the predicted secondary structure elements & predicted dihedral angles (labeled with Good and in green) extracted from the NMR data with TALOS to the ones of the crystal structure.