Spectrum Assignment using Ansig Ansig Setup

Transcription

1 Spectrum Assignment using Ansig Ansig Setup First you will go through the backbone assignment procedure. Go into the ansig directory. This contains several files: 1. The embo_back.ctr defines the al procedure directories (lib and user_lib), the amino acid dictionary, the sequence file, the spectrum description file, the crosspeaks file, the crosspeaks backup and the initialisation file. 2. The sequence (werner.seq) just consists of the residue numbers and names. 3. The embo_back.spd (spectrum description) file contains a list of the spectra you will be using. Have a look at this - the information it contains should be self-explanatory. 4. The crosspeaks file embo.crp is a binary - crosspeaks files can be output as text by typing crosspeaks export filename.xpk within ansig. A crosspeak export file can be imported into ansig (using crosspeak import) but this action removes any crosspeaks you already have. 5. The initialisation file embo_back.ini compiles all the AL procedures and macros. At the end it fires up all the graphics windows. It looks very complicated, so at this point it is best to see what Ansig can do and then relate back to the ini file. Starting up Ansig: Start up Ansig by typing: ansig embo_back.ctr it runs through the ini file and compiles the macros, loads the crosspeaks and brings up 6 windows. Now you are in text mode. To go to graphics mode, type g and return.

2 Graphics and Windows First familiarize yourself with the graphics mode. There are lots of commands which you can click on to activate them. In Ansig clicking is exclusively done with the middle mouse button. On the left are four columns of intrinsic ansig commands. You will use some of these as you go along. Next is a list of the spectra you have loaded. Clicking on the name of a spectrum will give you some information. Moving to the right, there are commands to manually assign and deassign crosspeaks. Next is the sequence - click on Met (the first residue). This should give you a list of all the Met residues, their surrounding residues in the sequence and the random coil shifts of Met atoms. At the moment, all Met residues are unassigned (you haven t made any assignments yet). Next to the sequence is a scroll bar - this allows you to scroll through the sequence. The two columns on the right are user-defined macros. The graphics window that you are working in has the word current in the top right corner. To change to a different window click in it (with any button). The size and positions of the windows as they are now was set up by the embo_back.ini file, but you can change them just like any other window. If you change the size, the current window position is printed out in the text window. In the left window (w1), the contours of the HSQC spectra are displayed. Zoom in and out by holding down the right mouse button and pushing the mouse forwards and backwards. Move the spectrum around by holding down the left mouse button and pushing the mouse around. The red line at the side of each window tells you how much of the third dimension is being displayed right now. Since you haven t loaded any contours you can t see any data. If you go to w4 and zoom in, you should see lots of crosspeaks - these have been picked in the triple resonance experiments. To display one plane of the 3D at a time, pick up the top of the red scroll bar at the side of the window, with the middle button of the mouse and pull down. This 2

3 displays gradually less of the spectrum. The scroll bar can be pushed around with the left button once it is narrow. As a start, the peaks in the 15n_hsqc and the backbone experiments have been picked - the crosspeaks appear as crosses. The 15n_hsqc_c is the HN/15N projection of the HNCA. If you look at the two HSQCs, you will see that there are some slight differences in the peak positions. There is no need to peak-pick the 15n_hsqc_c, just have it there for reference. To toggle on/off any of the spectra, click toggle in the intrinsic ansig commands and then the spectrum name. Alternatively hit the tab key, followed by the number in the list (e.g. to switch off the 15n_hsqc, tab 1). This command is often useful when you are looking at lots of spectra together. The default situation in Ansig is to display all spectra in every window where the dimensions of the window match the nucleus of the experiment. In w2 and w3 the 15n_noesy and hccconh will be displayed; in w4, w5, and w6 the hnca, cbcaconh, cbcanh and ccconh are displayed. Looking at 3Ds Click show_hh1 then click a peak in the HSQC (if you click close to a peak it is good enough to select it, if there are two peaks together, there will be an error unless you can pick one unequivocally). w2 will jump to the correct plane in the 15n_noesy and hccconh. To figure out which is which toggle the spectra on and off: The 15n-noesy is red and the hccconh is yellow. Now it is hard to see which peaks in the 3D correspond to your HSQC peak, so mark the crosspeak you have just clicked - to do this, click mark (or press m) and then click the crosspeak. You can look more closely at the strip in the 3Ds by zooming and pushing the spectrum around in the window. If you have two peaks that are overlapped in the NH but separated in the 15N dimension, you might want to look at the 15N/1H plane. To do this, click on show_n_noe and then the same HSQC crosspeak - it 3

4 should appear in w3, and you can zoom and push it around in the same way. To look at the triple resonance experiments: the corresponding commands are show_cacb and show_cacb2 which show the NH/13C plane in windows w4 and w5. The 15N/13C plane is displayed by show_cn in window w6. The four spectra you see when you use these macros are all different colours, as are their crosspeaks. The HNCA is yellow, the CBCA(CO)NH is green, the CBCANH is cyan and pink and the CCC(CO)NH is blue. Procedure for sequential assignment. To save time and let you have an idea of how things work, I have picked the peaks in the backbone experiments as follows: in the HNCA only the intra-residue CA correlation is picked; in the CBCA(CO)NH both CA and CB inter-residue correlations are picked; in the CBCANH the intra-residue CB correlation is picked; in the CC(CO)NH the inter-residue CG, CD, CE etc. correlations are picked. Go to crosspeak number 31 in the HSQC - either click mark (or press m) then type cp31 or look for the peak at (15N), 8.24 (1H). Jump to the correct plane in the triple resonance experiments (with show_cacb) for this peak and look at the positions of the peaks in all the spectra. What residue types might be this residue and the preceding residue? The correlation map might help. Now you want to move forwards and/or backwards in the sequence to try to make the sequential assignment. To move backwards, you want to look at all residues that have an HNCA peak at ppm and a CBCANH peak at To move forwards you want to look 4

5 for all residues that have CBCA(CO)NH peaks at the positions of the CA and CB peaks of this residue (57.24 and ppm). Click on close_in_c, then click on the CBCA(CO)NH peak at and zoom in to that region. Now it will ask you for a spectrum whose crosspeaks will be examined for a match in this case we want to find HNCA peaks that line up, so click on the spectrum name hnca. The program returns two possibilities ( 2 matching crosspeaks found ). The first possibility is shown in window w5 and the crosspeak is marked. To look at the other possibility type n, to go back to the first type p and to get out of this list type q. Work out which of these two possibilities is correct, based on all the data that you have in the triple resonance experiments. You might find it helpful to use the rulers - these are the solid white lines - to move them pick them up with the middle button. They are connected amongst the windows, so if you move the ruler to the CB shift in window w4 it will appear at the same place in window w5. Once you have decided on one of the two possibilities, use the same procedure to go forwards in the sequence, this time using the HNCA peak from crosspeak 31 at ppm and looking for matches in the CBCA(CO)NH that also line up with the CBCANH peak at ppm. There should be 3 possibilities for the CA match but only one matches CA and CB. Now that you have 3 residue types can you fit them into the sequence? Assigning Crosspeaks To actually assign a set of crosspeaks is somewhat tedious, so you can use some shortcuts. There are 3 assignment fields for an Ansig crosspeak: residue number; amino acid; nucleus. Since you have the sequence loaded, If you assign the residue number the amino acid is automatically assigned. To assign all dimensions of a crosspeak to be the same sequence click ass_seq : when it asks for a sequence number, either click on the sequence number or type in the number. 5

6 Do this for the HSQC peak. Now assign the nuclei in both dimensions: one way is to click ass_nuc, then click the crosspeak and answer yes or no to the possibilities it gives you. Now click correspond (or type o ) and click the HSQC peak followed by the HNCA peak. This propagates the NH assignments into the HNCA. Now assign the third dimension: click set_same_seq then the crosspeak and the sequence will be assigned. Assign the nucleus using ass_nuc again. To carry the assignment of the NH through all the other backbone experiments connect the crosspeaks. Connecting will only work if you have at least one dimension that is different so it will not be possible to connect two peaks that are on top of each other. To connect click connect or type c and then the two crosspeaks. The assignments of 15N and NH will be propagated and lines will appear between the connected peaks. Once a crosspeak has been assigned, it can no longer be moved, deleted or unaliased in the normal way. To delete it, it must be cleaned first. Sidechain Assignment The sidechain and backbone setups use the same crosspeak file so all of the assignments that you have made so far will be carried over. First, exit from the current (backbone) Ansig session by clicking tty and typing quit. Start up again with the sidechain control file (embo_side.ctr). A different set of windows will appear which you might need to move around a bit. In w1 is the 15N HSQC. w2 shows the 1H/1H planes of the 15N-NOESY and HCC(CO)NH spectra, w3 the CBCA(CO)NH and CBCANH spectra and the 13C HSQC. w4 and w5 show 1H/1H planes of the HCCH TOCSY and 13C-separated NOESY; w6 shows 13C vs. the indirect 1H dimension of the HCCH TOCSY and 13Cseparated NOESY. 6

7 Now that you have brought in new spectra to the display, some of the backbone experiments are no longer displayed. Now you are only looking at the cbcanh and the ccconh (which between them should contain all the information you want). The first thing to do is to copy the crosspeaks from the hnca and the cbcaconh onto the cbcanh and ccconh respectively. To do this, click cp_copy then type in hnca and click cbcanh. Click cp_copy again then type in cbcaconh then click ccconh. To display all the crosspeaks you have assigned in the 15N-HSQC: click assignments, then the spectrum name. Start at Glu-67: load the 1H/1H plane of the 15n_noesy and the hccconh experiments. Now you will peak-pick the hcconnh experiment to get the 1H shifts of Val- 66. Peak-peaking There is no intrinsic peak-picking routine in Ansig, instead you will use a macro that creates an input file for the Azara program peak_find, runs it and brings back the cross-peaks. This works very efficiently for picking 2D and 3D spectra. Like most peak-picking programs, peak_find will pick noise as well, so it is usually best to select a small region of the spectrum of interest. To zoom in on the region of interest (in this case the crosspeaks in the hccconh), click current, then box - zoom out a little way and you will see a box around the peaks. It can be resized by moving the sides with the mouse (middle button). When you are happy with it click yes, then peak_find. It will ask you for a spectrum - select the hccconh. Peak_find will run and finally tell you how many peaks it has found. Remove any spurious peaks. There is also manual picking in graphics mode - click pick, followed by the spectrum, then click in the position where you want the peak. Once the Val-66 1H peaks have been picked and you have assigned them in the hccconh, bring up the Val-66 or Glu-67 cbcanh and ccconh planes. To see the assignments you have made so far for these residues, click show_ss_all then residue number. Click 7

8 show_chh1, then the crosspeak corresponding to the CA of Val-66 - window w4 will jump to the CA plane in the hccht and cnoe experiments. Here you can pick and assign the α/β, α/γ1 and α/γ2 crosspeaks for Val-66. To check that the peaks are not overlapped in the 13C dimension, click show_ch_c, then the picked peak in the hccht. This will display, in window w6, the 13C vs. 1H plane at the chemical shift of the HA. To look for the β/α crosspeak at the CB position, click show_hh_c then the α/β crosspeak. This will bring up, in window w6, the 13C vs. 1H plane at the 1H shift of the HB. If you mark the α/β crosspeak in the CA plane, you will look for the symmetry related peak in the CB plane. In this case you know it is at ppm, so you can find it easily. It might be easier to see if you toggle off the 13C-separated NOESY. Using the same procedure you should be able to find the rest of the Val-66 spin system. 8