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NMR wisdom:Protein NMR - A Practical Guide - Solution NMR Assignment - Practice
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[/B]Assignment - Practice (for Analysis 2.1)
 This section describes how the assignment principles described under [URL="http://www.protein-nmr.org.uk/assignment_theory.html"]Assignment - Theory[/URL] can be but put into practice using the [URL="http://www.ccpn.ac.uk/ccpn/software/ccpnmr-analysis"]CCCPNmr Analysis[/URL] software. There are several ways in which triple resonance backbone assignment, in particular, can be approached in [URL="http://www.ccpn.ac.uk/ccpn/software/ccpnmr-analysis"]CCPNmr Analysis[/URL]  using more or less automated methods. Initially a more manual method  will be described, as this makes it easier to understand the process of  assignment for those who are new to protein NMR assignment. This is  followed by an outline of the slightly more automated method using the [B]Link Sequential Spin Systems[/B] function within [URL="http://www.ccpn.ac.uk/ccpn/software/ccpnmr-analysis"]CCPNmr Analysis[/URL]. 
  [B]This section describes assignment using  Analysis 2.1. If you are using an older version, then have a look at the  old webpages for [URL="http://www.protein-nmr.org.uk/assignment_practice_v1.html"]version 1.0[/URL] or [URL="http://www.protein-nmr.org.uk/assignment_practice_v20.html"]version 2.0[/URL] for how to do the assignment, as some things have changed between versions.[/B]
  This section assumes that you have created a new project, provided details of the [B]Molecular System[/B] and read in your spectra. See [URL="http://www.protein-nmr.org.uk/analysis2.html"]CCPNmrAnalysis Basics[/URL]  for details on how to do this, as well as how to change the appearance  of your spectra and windows, use the mouse and navigate around your  spectra.
 [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#top"]Top[/URL]
    Initialising the HSQC
 By initialising the HSQC you select all its peaks and then create new  resonances for each peak. In addition, resonances from backbone peaks  are grouped into spin systems and their atom types are  also assigned.
  Begin by peakpicking your HSQC. The easiest way to do this is to zoom  out at a fairly high contour level and then drag the mouse over all  peaks while holding down [B]Shift[/B] and [B]Ctrl[/B]. Avoid picking too many artifacts by making sure that only positive peaks can be picked (this option can be selected under [B]Peak Finding[/B] in the [B]Peaks[/B]  menu). You will probably still find that some small positive artifact  peaks have been picked which you do not want - delete these by selecting  them and pressing [B]Delete[/B]. There will  also be peaks which have not been picked correctly because of overlap -  simply set a peak at the place where you want it by [B]Right Clicking[/B] the mouse and selecting [B]Peak[/B] and then [B]Add new peak[/B].
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/hsqc_peakpick_text.png[/IMG]
  Once you are happy that you have picked all the peaks you want, go to [B]Initialise Roots[/B] in the [B]Assignment[/B] menu. You should make sure that the correct peak list is selected in this pop-up (top left corner). When you click [B]Initialise Peak List![/B]  Analysis will assign two resonances to each peak - one in each  dimension (resonance numbers are always surrounded by square brackets  []). Because Analysis knows that this is an HSQC, it will also create a  spin system for each peak, and both resonances from the peak will be  added to it (spin system numbers are always surrounded by curly brackets  {}). Furthermore, the hydrogen resonances will all be assigned the atom  type [B]HN[/B] and the nitrogen resonances the atom type [B]N[/B].  While this is the correct way to treat the backbone peaks in an HSQC,  this is not the correct treatment for side-chain peaks, in particular  the Asn and Gln side-chain peaks. Here the atom types are different ([B]Hδ[/B], [B]Hε[/B], [B]Nδ[/B] and [B]Nε[/B] instead of [B]HN[/B] and [B]N[/B]) and because we are dealing with NH2  groups, there will be two peaks which belong to the same spin-system  and indeed will have the same resonance in the nitrogen dimension.  Analysis therefore helps you identify the Asn and Gln side-chain peaks,  so as initialise them seperately, and only assign resonances to them,  not spin systems and atom types.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/hsqc_initialise_popup.png[/IMG]
  The [B]Amide Side Chain Peaks[/B] table in the [B]Initialise Roots[/B]  pop-up lists all the pairs of peaks that Analysis can find which have a  nitrogen chemical shift within a certain tolerence of one another. The  size of this tolerance can be set in the top right hand box of the [B]Initialise Roots[/B] pop-up. When you click on one of the rows in the [B]Amide Side Chain Peaks[/B]  table, Anaylsis will automatically navigate to that pair of peaks and  mark them. The window used for this can be selected from the drop-down  menu in the top part of the pop-up.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/hsqc_initialise_nq.png[/IMG]
  If a given row has (in your opinion) correctly found an amide side-chain peak pair, then either click [B]Confirm Selected[/B] or double-click the cell in the [B]Confirmed?[/B] column. This will now switch from [B]No[/B] to [B]Yes[/B].  Check all the rows. If this has not identified all the amide side-chain  peaks, then increase the tolerance (top right hand corner of the  pop-up) and click [B]Refresh Table[/B] to  update it until all pairs are picked up. Once all amide side-chain peaks  are confirmed, the initialisation can be done by clicking [B]Initilise Peak List![/B].
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/hsqc_initialised1_text.png[/IMG]
   [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#top"]Top[/URL]
   Peakpicking the 3D spectra
 All the peaks in a 3D spectrum should in theory be arranged in  columns above the peaks of the 2D root spectrum it derives from (see [URL="http://www.protein-nmr.org.uk/assignment_theory.html#vis3d"]Visualising 3D spectra[/URL]  for an explanation). Therefore the fastest way of peak-picking a 3D  (and also the best way of avoiding picking too much noise) is to pick  peaks only in the columns above the peaks you have in your 2D root  spectrum. So if, for instance, you are wanting to peak-pick an [URL="http://www.protein-nmr.org.uk/spectra.html#hnco"]HNCO[/URL] or a [URL="http://www.protein-nmr.org.uk/spectra.html#cbcaconnh"]CBCACONNH[/URL] spectrum, you would base it on the peaks in your [URL="http://www.protein-nmr.org.uk/spectra.html#hsqc"]HSQC[/URL]  spectrum. Analysis is then able not only to do the peak picking, but  will also assign the nitrogen and hydrogen resonances and atom types  from the root HSQC peaks to CBCACONNH peaks.
  Before you peakpick each 3D spectrum set your peak picking parameters in the [B]Peaks[/B] / [B]Peak Finding[/B] pop-up. Make sure that you have selected [B]positive and negative[/B] or [B]positive only[/B] depending on the peaks that you want to pick in your 3D. [B]Scale relative to contour levels[/B]  is probably set to 1.0, so you should now set your contour level such,  that you only see (and therefore pick) real peaks and not noise. Later  you may want to lower the contour level again, but for the main peak  picking operation it is usually useful to have the contour level set  relatively high.
  To peakpick your 3D CBCACONNH spectrum go to the [B]Assignment[/B] menu and select [B]Pick & Assign From Roots[/B]. At the top of the [B]Navigation Windows[/B]  tab you need to select your root peak list (i.e. probably your HSQC  peak list) and the window in which you want your root spectrum to be  displayed. At the bottom of the tab you can select from a drop-down menu  the window in which you want to display your 3D spectrum which you are  going to peakpick. Now click on [B]Add Window[/B] to add this to the list above.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/pickassign_roots_nav_text.png[/IMG]
  Now move to the [B]Spectra & Tolerances[/B] tab. This will show all the spectra which are visible in the window you selected in the [B]Navigation Windows[/B] tab. Enter suitable tolerances at the top of the tab (something like about 0.02, 0.1 and 0.2 for 1H, 15N and 13C,  respectively, should be reasonable starting values). If your 3D  contains a water peak or a diagonal (e.g. if picking a NOESY spectrum),  then you can exclude these regions using the other tolerance boxes.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/pickassign_roots_tol_text.png[/IMG]
  In the [B]Link Peaks[/B] tab you should now  get a list of all the spin systems in your root peak list. If you click  on one, then Analysis will automatically navigate to its position in  the root and 3D spectra in the windows selected previously. When you  click on [B]Pick & Assign Root Resonances[/B]  Analysis will automatically pick the peaks in the 3D and assign the  resonances belonging to the root dimension. To start with it may be  worth going through some of the spin systems manually in this way in  order to check that your tolerances and contour levels seem reasonable.  Once you think that you have got these levels about right, you can also  get Analysis to peak pick and assign resonances for all spin systems in  one go by clicking on [B]Pick All & Assign Root Resonances[/B]  at the centre bottom of the pop-up. Although this is quicker intially,  it can sometimes pay off to go through each spin system individually.  Analysis will inevitably make mistakes (see below). You can of course  correct these mistakes as you come across them during the assignment  process, but often the mistakes can actually slow down your assigning  and so you may decide that you would rather go through each spin system  one at a time and then be able to start off with a nice tidy set of  peaks in your 3D.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/pickassign_roots_peaks_text.png[/IMG]
  When the peak picking is done you will find that the atom type  information as well as the resonance and spin system numbers have been  transferred from the HSQC onto the 3D. This means you are only left with  having to assign the carbon dimension of the 3D peaks which will speed  things up considerably.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/peakpick_3d_assigned_short2_text.png[/IMG]
  You will almost certainly find that some strips will be stronger than  others. Even when you have set all the peak picking parameters as  sensibly as you can, you are likely to end up with some strips that  contain a large degree of noise and therefore additional peaks have been  picked. Simply delete such peaks when you come across them.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/peakpick_3d_noise_short_text.png[/IMG]
  There may be cases where two peaks are very close to one another in  the HSQC and within the tolerances you set for the 3D peak picking. In  this case you will probably find that that all 3D peaks have been  assigned the same resonance and spin system information. If you come  across such strips in your 3D, you will need to compare the spectrum  carefully with your HSQC and work out which 3D peak belongs to which  root peak in the HSQC. Then the assignments on the 3D peak will need to  be changed.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/peakpick_3d_overlap_text.png[/IMG]
 In order to do this, you will need to call up the assignment panel. Simply place the mouse over the required peak and press [B]a[/B]. This way the assignment panel will immediately show the assignment for that peak.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_panel_text.png[/IMG]
 To change the assignment, in this case from resonances {72}H[143] and  {72}N[144] to {155}H[313] and {155}N[314], select the {72}H[143]  resonance on the left and click [B]Clear Contrib[/B]  to remove it from that peak. The new resonance {155}H[313] can be  assigned to the peak by clicking on it on the right hand side of the  panel. The same process will then have to be repeated for the nitrogen  dimension.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_panel_selection_text.png[/IMG]
  Once you are happy with the peak picking for each 3D spectrum, save your project and peak pick the next spectrum. 
  [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#top"]Top[/URL]
   Triple Resonance Backbone Assignment
 The description here assumes that the backbone assignment will be  carried out using CBCA(CO)NH and CBCANH spectra. Many of the steps are  the same if using HNCA, HN(CO)CA, HNCO and HN(CA)CO spectra, and  differences are highlighted in a section below.   
Protein Sequence Assignment - Manual
 Now comes the actual assignment part which follows the process outlined in the [URL="http://www.protein-nmr.org.uk/assignment_theory.html#tripbb"]theory section[/URL].  This section shows how to do this manually and in so doing goes through  each step one at a time. Once you feel comfortable with the general  process you can speed things up by using the semi-automated [B]Protein Sequence Assignment[/B]  function. This simply condences several steps into one, thus speeding  the process up. The flow diagram below shows the different steps that  need to be taken.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_flow_diagram.png[/IMG]
  [B][I]1. Find a peak in the HSQC to be used as a starting point[/I][/B] 
Begin by selecting a peak in the HSQC which looks nice and is not overlapped. 
  [B][I]2. Navigate to this NH position in the CBCANH[/I][/B] 
Navigate from here to your CBCANH by [B]right clicking[/B] the mouse and selecting [B]Navigate[/B] and [B]1H - 15N in windowX[/B] (where windowX is the window in which you have the CBCANH). The strip should contain two Cα peaks and two Cβ peaks.   
[B][I]3. Identify the own and previous Cα/Cβ pairs by comparison with the CBCA(CO)NH[/I][/B] 
If you overlay the CBCA(CO)NH, then one of the Cα and one of the Cβ  peaks (generally the weaker ones) should be present in the CBCA(CO)NH.  These are the Cα and Cβ of the previous residue, the other two are the  Cα and Cβ of the same residue as the NH group. The two carbons belonging  to the same residue as the NH group can be assigned.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/matching_hsqc_cbcannh_text.png[/IMG]
  [B][I]4. Assign the own Cα/Cβ[/I][/B] 
The assignment is done using the assignment panel (press [B]a[/B] when the mouse is over the peak to be assigned). Select [B]New[/B]  for the carbon dimension. This will create a new carbon resonance and  assign it to this peak. Since it belongs to the same spin system as the N  and H resonances, click on [B]Set Same Spin System[/B], so as to add the new carbon resonance the NH spin system. Now assign the atom type (Cα or Cβ) by clicking [B]Set Atom Type[/B]. This will bring up the [B]Browse Atoms[/B] panel which will initially not show any atoms at all. Click on [B]C[/B] to toggle the carbon atoms on and select any [B]Ca[/B] (or [B]Cb[/B]) to set the atom type.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_atoms_panels_text.png[/IMG]
  [B][I]5. Select the own Cα/Cβ and search for matching peak pairs in the CBCA(CO)NH[/I][/B] 
Now select your assigned Cα and Cβ peaks (drag the mouse over them together or individually while holding down [B]Shift[/B]) and to make things easier, place a mark through each peak (hold the mouse of the peak and press [B]m[/B]). Then hold the mouse over one of the two peaks, [B]right click[/B] and select [B]Peak[/B], [B]Match Peaks[/B], [B]In CBCACONH[/B] and [B]F3[/B]  (make sure this is the carbon dimension!). Analysis will now look for  strips in the CBCA(CO)NH spectrum where there are peaks that match these  two carbon dimensions. In this way you will find the NH which follows  your current one in the sequence, since the CBCA(CO)NH peaks are of the  type Hi-Ni-Cαi-1/Cβi-1. Be  aware that at the peak matching feature will only offer those spectra  which are visible in your query window as possible options for matching!  So if the spectrum in which you want to search for matches is not given  as an option, make sure that it is 'switched on' and visible in the  current window from where you are doing your matching.
 Analysis will then bring up a new panel in which the Options are  presented. The case below two matches were found which are ranked  according to a scoring function. The top match has matches to both  peaks, the lower one only to one peak. Thus the top match looks as  though it is probably the correct one, though this should always be  checked visually in the spectra as well (a peak found by analysis may  for instance be noise and not a real signal, or if two peaks are  overlapped the peak may not be placed correctly). 
  [B][I]6. Display matching peaks in strips and select best match[/I][/B] 
To visually inspect your results, you can select as many of the possible matches as you like. [B]Diplay Groups in Strips[/B] will then bring up these strips in whichever target window you have selected.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_matched_peaks_text.png[/IMG]
  Once you have brought up your possible matches for visual inspection  you may be lucky and find that there is quite definitely one match which  is much better than the others. 
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_matched_peak_strips_orig_text.png[/IMG]
  [B][I]7a. Good match: Assign the CBCA(CO)NH Cα/Cβ and set the NH as the sequential spin system[/I][/B] 
In this case you can now go ahead and assign the carbon dimensions  in the matching strip of the CBCA(CO)NNH. Simply bring up the [B]Assignment Panel[/B] for each peak (press [B]m[/B] while the mouse is on the peak) and select the relevant carbon resonance.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/assignment_panel_matched_peak_text.png[/IMG]
  To set the sequential spin system, select either peak, [B]right click[/B] the mouse and go to [B]Assign[/B], [B]Set Sequential Spin Systems[/B], [B]F1 0, F2 0, F3 -1[/B].  Analysis now knows that the spin system assigned to the F3 dimension of  this peak belongs to the residue i-1 relative to the spin system  assigned to the F1 and F2 dimensions.
 Sometimes you will find tha there are two very good matches and it is  not possible to tell which is correct. In this case simply make a note  of the spin system involved and come back to this later when you have an  idea of the position in the sequence and the amino acid types that are  involved.
  [B][I]7b. No good match[/I][/B] 
If you are unlucky and the spectra are not very good, you may find  that none of the possible matches found by Analysis match very well.  Alternatively, the following amino acid may be a proline - because it  has no H attached to its N, proline does not give any signals in  CBCA(CO)NH spectra and there is no possible match to find. If cannot  find a good match, simply make a note of the spin system you have  investigated and try your luck with another by starting at the beginning  again.
   Protein Sequence Assignment - Semi-Automated
  To use the [B]Protein Sequence Assignment[/B] function, select this from the [B]Assignment[/B] menu and the [B]Protein Sequence Assignment[/B]  panel will come up. First of all you need to select the window and  spectrum from which you do your search, i.e. the CBCANH. Then select the  window in which you want the possible matches to be displayed and the  spectrum in which you want to find your matches, i.e. the CBCA(CO)NH.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/protseqassign_settings_text.png[/IMG]
  In the [B]Spin Systems Table[/B] tab you  will then find a list of all your spin systems at the top, then a list  of possible matches for each spin system and in the bottom part of the  panel, there are some indictors of the amino acid type and possible  sequence position.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/protseqassign_table_text.png[/IMG]
  When you select a spin system in the top table, Analysis will  automatically navigate to this position in your query spectrum (CBCANH)  in the window specified previously. It will also immediately look for  matches to the peaks in that strip. These are then listed in the [B]Matched Peak Positions[/B] box and are displayed as strips in the other window selected previously.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/linkseqspinsys_strips_text.png[/IMG]
  First of all go to the CBCANH and assign the Cα and Cβ resonances  which belong to that strip's own spin system (i.e. the strong peaks  which do not overlap with the CBCA(CO)NH). Simply press [B]a[/B] when holding the mouse over each peak, add a [B]New[/B] carbon resonance, then [B]Set Same Spin System[/B] and [B]Assign Atom Type[/B].
 Now you can check whether any of the matches found in the CBCA(CO)NH  fit to this resonance. In the example case, spin system {54} is the only  one which matches and must therefore be the one which follows {88} in  the sequence. In order to assign the carbon dimension of the {54}  CBCA(CO)NH peaks and set it as the spin system following {88}, simply  select the {54} row in the [B]Matched Peak Positions[/B] box and click [B]Set Seq Link[/B].
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/protseqassign_table_linked_text.png[/IMG]
  Now you can move on to the next spin system. Simply click [B]Goto i+1[/B]  and Analysis will now set spin system {54} as the starting spin system  from which to look for matches. It immediately navigates to strip {54}  in the CBCANH and shows the CBCA(CO)NH matches. All you have to do is  assign the {54} Cα and Cβ, select the match and move on. Simple! :)
  If you cannot find a good match, or there are several that match  equally well, just make a note of this and move on to another spin  system (e.g. with [B]Goto Next[/B]). Note that in the [B]Settings[/B]  tab, you can experiment with setting your Tolerance to be larger or  smaller or with the number of strips to be displayed in the CBCA(CO)NH  window.
  If you find that you have made a mistake at some point, simply select the spin system in question in the [B]Spin Systems[/B] box and click [B]Clear i-1 links[/B], [B]Clear i+1 links[/B] or [B]Clear all links[/B] and then you can set new links again.
   Sequence-Specific Assignment
 By linking sequential spin systems you will have ended up with a  number of long strings of spin systems in their correct sequential  order. Now you need to work out which part of the sequence they match up  to. In order to do this you need to identify the amino acid type of  some of your spin systems. For some amino acid types this is fairly  straight forward. Glycine for instance, has a very characteristic Cα and  no Cβ chemical shift. Alanine, Serine and Threonine all have very  characteristc Cα and Cβ chemical shifts. You can see the distributions  of chemical shifts by amino acid type as found in the [URL="http://www.bmrb.wisc.edu/"]BMRB[/URL] within Analysis if you go to [B]Resonance[/B] and [B]Reference Chemical Shifts[/B]. The following [URL="http://www.protein-nmr.org.uk/chem_shift_table_carbon.pdf"]pdf file[/URL]  has a visually very appealing way of displaying the average carbon  chemical shifts. Using this you can quickly see that Isoleucine, Proline  and Valine have lower Cα chemical shifts than all the other amino  acids, for example.
  Once you have identified (or excluded!) the amino acid type of a few  of your spin systems in your linked stretch, you can start to compare  this to your protein sequence. You may for instance have a stretch which  is S-X-A-X-X-G, where X is, for example, anything other than A, S, G, T  or P. If this motif only appears once in your sequence, then you can  make a sequence specific assignment. If you like, you can immediately  enter this into Analysis. But I tend to wait until I have assigned most  of the sequence and am feeling fairly confident with my assignment  before doing this, as it makes undoing mistakes easier. To enter your  sequence specific assignment in Analysis, simply bring up the [B]Assignment Panel[/B] for one of the peaks in one of the spin systems you want to assign. Select a resonance and click [B]Assign {SpinSystem}ATOM[Resonance][/B]. The [B]Atom Browser[/B] panel will appear and you now need to select the exact atom which this resonance corresponds to.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/seqspec_assignment_text.png[/IMG]
  This atom will then be shaded in a slightly darker colour in the [B]Atom Browser[/B]  panel to indicate that it is assigned. But you will find that there are  lots of other atoms which will become shaded at the same time, because  Analysis is able to assign them based on the assignment you have just  made. For instance, if you assign the Cβ in one spin system, then  Analysis will automatically be able to sequence-specifically assign the  rest of that spin system, too. And if there is another spin system which  has been set to be i-1 to this spin system, that is automatically  assigned, too. So with one click, you can set the sequence specific  assignment of a whole stretch of residues.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/browse_atoms_assigned_text.png[/IMG]
     Using HNCA/HNCO Spectra
 If you are using HNCA, HN(CO)CA, HN(CA)CO and HNCO spectra for your assignment the easiest thing is to use the [B]Protein Sequence Assignment[/B]  function in a mannner very similar to that described above. When using  the CBCANH/CBCA(CO)NH spectra the Cα and Cβ resonances are matched. When  using the HNCA and HNCO based spectra, it is the Cα and CO resonances  which are matched. Within the [B]Protein Sequence Assignment[/B]  panel you will have to set both the HNCA and HN(CA)CO as your query  spectra. In these spectra you will need to assign the Cα and CO  resonances belonging to the query NH group. The matched spectra in which  you look for your matches will be the HN(CO)CA and HNCO spectra. One  difficulty that arises when using the HNCA/HNCO spectra is that the CO  resonances have chemical shifts between about 170 and 180 ppm. So while  the Cα and Cβ resonances occur at similar ppm values (about 75-15 ppm)  and it is easy to look at these in the same window, this is not so easy  if you want to inspect the Cα and CO resonances at the same time. To get  around this problem, you can simply tick the [B]Focus 13C Matches[/B] option in the [B]Settings[/B] tab of the [B]Protein Sequence Assignment[/B] pop-up.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/protseqassign_panel_hncahnco_focusview_text.png[/IMG]
  Analysis will then automatically add horizontal separators to your  windows and focus the window sections on all the relevant peaks. This  means that you can look at the HNCA and HNCO spectra in the same window  rather than having to juggle additional windows or do lots of scrolling.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/linkseqspinsys_strips_hncahnco_focusview_text.png[/IMG]
  Now you can select a spin system in the [B]Protein Sequence Assignment[/B] panel, and match it in the normal way.
  [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#top"]Top[/URL]
     Double Resonance Backbone Assignment
 Assigning proteins which are only 15N-labelled is harder than assigning 15N,13C-labelled  proteins. But for proteins of up to about 130 residues in length this  is not an unreasonable task, especially, if the secondary structure and  topology are known from a crystal structure or a homologous protein. The  general principle of assignment is outlined in the [URL="http://www.protein-nmr.org.uk/assignment_theory.html#doubbb"]theory section[/URL]. In addition to your [URL="http://www.protein-nmr.org.uk/spectra.html#hsqc"]HSQC[/URL] spectrum you will need a 3D [URL="http://www.protein-nmr.org.uk/spectra.html#15nnoesy"]15N-NOESY-HSQC[/URL] and a 3D [URL="http://www.protein-nmr.org.uk/spectra.html#15ntocsy"]15N-TOCSY-HSQC[/URL].  There is no set way of doing double resonance backbone assignment in  Analysis, but several useful moves and strategies are outlined below. It  is assumed that you have already [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#inithsqc"]initialised your HSQC[/URL] and [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#peakpick3d"]peakpicked your 3D spectra[/URL].
  The overall workflow is as follows:   
(a) [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#neighb"]Find the sequential neighbours[/URL] to each residue using the NOEs between their NH groups. You can do this relatively easily using the [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#matchpeaks"]Match Peaks[/URL] function. 
(b) [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#direct"]Determine the correct sequential order, or directionality[/URL], of the neighbouring residues using NOEs between the NH group of one residue and the side-chain of the other. 
(c) Once you have strung several residues together sequentially,  identify (or exclude) the amino acid type of some of the residues and  match this to your sequence in order to obtain the [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#seqspecassign"]sequence-specific assignment[/URL].
 If you know the secondary structure and topology of your protein, you  can use this to help you. α-helices and β-sheets have slightly  different patterns of NOEs which can help with the assignment - residues  with α-helical or β-sheet NOE patterns will necessarily be grouped  together in the sequence. If you have a β-sheet, then the cross-strand  NOEs can help you assign and build up two neighbouring strands in  parallel.
   Neighbouring Residues
 In your 15N-NOESY-HSQC spectrum you should be able to see NOEs  between neighbouring NH groups. There are several ways of identifying  neighbouring residues. To begin with, select a peak in your HSQC with  which you want to start and navigate to that position in the  15N-NOESY-HSQC ([B]right-click[/B] the mouse and go to [B]Navigate[/B] and [B]windowX[/B] where windowX is the window in which you have your 15N-NOESY-HSQC).
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_hsqc_noesy_nav_text.png[/IMG]
   With any luck there should be two or more NOEs in this strip to other  NH groups. Two should be to the neighbouring residues (i.e. NOEs to  NH(i±1)) and additional ones will be medium or long-range NOEs. In  α-helical secondary structure you may well see NOEs to NH(i±2) and  NH(i±3). These will be weaker and can be helpful in linking up a several  sequential residues. In β-sheet secondary structure you will see  long-range NOEs to the neighbouring strand which may be of comparable  strength to the NH(i±1) NOEs (parallel β-sheet) or considerably stronger  than the NH(i±1) NOEs (antiparallel β-sheet).
  In principle you want to find the correct spin system that each NOE  originates from. In some cases it may be possible to identify the spin  system unambiguously because the hydrogen chemical shift is unique to  one spin system. However, in many cases there will be several options.  One way to know that you have found the correct spin system, is by  looking for the symmetrical NOE peak. If spin system [I]i[/I] has an NOE to spin system [I]j[/I], then [I]j[/I] should also have one to [I]i[/I]. When you place the strips alongside one another this makes a nice rectangular pattern.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_strips_text.png[/IMG]
  Note that in the region between about 6.5 and 7.5 ppm you can not  only observe NOEs from NH groups, but also from certain side chains such  as Phe, Tyr, Trp, Asn or Gln. If your protein contains Trp residues,  you may also be able to observe NOEs to the Trp side-chain Hε1 nuclei  between about 9.0 and 11.0 ppm. The Asn, Gln and Trp Hε1 resonances are  also visible in your HSQC, however the Phe, Tyr and other Trp resonances  are not.
  It can be difficult working out which NOEs are from the neighbouring  residues and which are from others. And of course when you have found  out which are the neighbouring residues, you then still need to  determine which is on the N-terminal side and which on the C-terminal  side of your central residue. You can do this as outlined [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#direct"]below[/URL] using some of the side-chain NOEs. 
On the whole the NOEs to neighbours will be the stronger ones, and  they should be of comparable strength to one another. The main exception  to this, is the cross-strand NOE in an antiparallel β-sheet which is  stronger than the N(i±1) NOEs. NOE intensity scales with the distance  between the hydrogen atoms (as r-6). Below are some typical distances found in seconary structure (taken from Wüthrich (1986) [I]NMR of Proteins and Nucleic Acids[/I], WileyBlackwell).
                  
i, i ± 1 i, i ± 2 i, j  α-helix 2.8 Å 4.2 Å 
 β-sheet (parallel) 4.3 Å 
4.8 Å  β-sheet (antiparallel) 4.2 Å 
3.3 Å    You have several options of how to identify which are your  neighbouring strips. Probably the easist option initially is to use the [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#matchpeaks"]Match Peaks[/URL] function. An alternative would be to select the assignment panel for each NOE peak (hold the mouse over the peak and press [B]a[/B])  and then make a note of each of the spin systems that is an option for  that NOE. Then you can line their strips up using the precedure outlined  [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#linestrips"]below[/URL] and look at the matches.
    Directionality
 When you have found two neighbours for a residue via the NH NOEs you  do not initially know which one is the N-terminal neighbour and which  the C-terminal neighbour. An easy trick is the fact that the Hα of  residue i is usually much closer to the NH of residue i+1, than the NH  of residue i-1. If you overlay your 15N-TOCSY-HSQC onto your  15N-NOESY-HSQC you can identify the Hα within the central strip (it will  be the first peak above the diagonal in the 15N-TOCSY-HSQC spectrum,  usually between about 4 and 6 ppm) and then you can see a  direction-dependent pattern: the strip belonging to the C-terminal  neighbour (i+1) should have a strong NOE to the Hα while the strip  belonging to the N-terminal neighbour (i-1) will have no or only a very  week NOE the Hα. This is particularly marked in β-sheet sections as you  can see below.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/directionality_betasheet_text.png[/IMG]
   Matching Peaks
 You can use the [B]Match Peaks[/B] function  in order to find possible neighbouring residues to your starting  residue. Simply select the diagonal peak of your starting residue in the  15N-NOESY-HSQC and the NOE you are interested in. Then [B]right-click[/B] the mouse over one of the peaks and go to [B]Locate Peaks[/B], [B]Match multiple peaks[/B], [B]In 15N_NOESY[/B], [B]F2[/B] (in older versions of analysis [B]right-click[/B] and go to [B]Peaks[/B], [B]Match Peaks[/B], ...). Note that the [B]15N_NOESY[/B]  part is the [B]Experiment Name[/B]  you gave your 15N-NOESY-HSQC and that the search dimension should be the indirect 1H dimension - this will usually be [B]F2[/B], but this may not always be the case - it depends on exactly how you recorded your experiment.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_matches_start_text.png[/IMG]
  Analysis will now look through the F2 dimension of your  15N-NOESY-HSQC peak list and look for matches to your query peaks. The  results are brought up in the [B]Group Peaks[/B]  pop-up. You will see a list of peaks sorted by a score - the higher the  score, the better the match. And of course you are interested in both  peaks matching, so results in which two peaks can be matched are better  than ones where only one matches. You can now select a group of peaks  that you would like to inspect visually. You can have them automatically  brought up as strips in a window of your choice. Make sure the [B]Target Window[/B] is the one you would like to use and then click on [B]Display Groups in Strips[/B].
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_matches_panel_text.png[/IMG]
  When investigating the strips (below) it is easily visible that only spin system {25} can be the correct match.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_matches_strips_text.png[/IMG]
  Make a note of the matching spin system, check for information on the [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#direct"]directionality[/URL]  ({19} has a strong NOE to the Hα of {25}, so {19} is probably  C-terminal to {25}) and then check for matches to the other NOEs in the  {19} strip.
 It is probably also worth assigning the NOESY peaks that link the two spin systems for future reference. Bring up the [B]Assignment Panel[/B] (hold the mouse over the peak in question and then press [B]a[/B]) and select the relevant resonance in the F2 dimension.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_assign_panel_text.png[/IMG]
  You can also set a sequential link between the two spin systems if  you are confident that they are neighbours and that you know the  directionality. Simply hold the mouse over the NOESY peak linking the  two spin systems and [B]right-click[/B] the mouse and go to [B]Assign[/B], [B]Set sequential spin systems[/B]  and select the option from the list which correctly describes which  dimension belongs to residue i +/- 1 relative to the other two  dimensions. Probably you will need to select either [B]F1 0, F2 -1, F3 0[/B] or [B]F1 0, F2 +1, F3 0[/B].
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/doubres_noesy_seqlink_text.png[/IMG]
  Lining up Strips
 Lining up strips of the 15N-TOCSY-HSQC or 15N-NOESY-HSQC can be  useful in a variety of contexts. Sometimes you may have noted down  several possible matches for a particular strip and want to look at  them. At other times you may think you have a string of sequential spin  systems and you may want to look at their strips in a row to review  whether this is correct or not.
 Begin by calling up the [B]Resonances[/B] pop-up from the [B]Resonances[/B] menu. Sort the resonances by [B]Spin System[/B] and then select the N and H resonances of your spin system of interest. Now make sure that the correct [B]Window[/B] which you would like to use is selected from the drop-down menu. Then click on [B]Go to Position[/B]  and the selected window will navigate to the NH position of the  selected resonance. If your target window contains several strips, then  it will be the active strip in that window which navigates to the new  position.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/resonances_navigate_text.png[/IMG]
  You can gradually build up a row of strips in your window. Either add  a new strip to your window or change the active strip (button  highlighted in green) and then go back to the [B]Resonances[/B]  pop-up in order to select your next HN resonance pair. You can also  delete or move your strips relative to one another using the buttons in  the [B]Strips Toolbar[/B] (see figure below).
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/lined_up_strips_text.png[/IMG]
   Sequence Specific Assignment
  Once you have linked a number of spin systems in their correct  sequential order you can start to work out which part of the sequence  they match up to. In order to do this you need to identify the amino  acid type of some of your spin systems. This is harder for double (1H, 15N) than for triple (1H,15N,13C) labelled protein, as the 1H and 15N chemical shifts are not as characteristic for their amino acid type as the 13C chemical shifts. But based mainly on the 1H  chemical shifts it is nonetheless possible to identify or exclude  certain types of amino acids for many spin systems. You can see the  distributions of chemical shifts by amino acid type as found in the [URL="http://www.bmrb.wisc.edu/"]BMRB[/URL] within Analysis if you go to [B]Resonance[/B] and [B]Reference Chemical Shifts[/B]. The following [URL="http://www.protein-nmr.org.uk/chem_shift_table_hydrogen.pdf"]pdf file[/URL]  has a visually very appealing way of displaying the average hydrogen  chemical shifts. Using this you can quickly see that Alanine, Glycine  and Threonine have unusual patterns of side-chain hydrogen chemical  shifts. The remainder can be devided into two groups: (1) those with  only Hα and Hβ chemical shifts in the 5-0 ppm range (Asn, Asp, Cys, His,  Phe, Ser, Trp, Tyr) and (2) those with longer side chains visible in  this region (Arg, Gln, Glu, Ile, Leu, Lys, Met, Val). However, always be  aware, that not all resonances may be visible in the 15N-TOCSY-HSQC,  especially from the ends of long side-chains, and that peaks may be  overlapped (especially the two hydrogens from the same CH2 group often have the same chemical shift).
  Once you have identified (or excluded!) the amino acid type of a few  of your spin systems in your linked stretch, you can start to compare  this to your protein sequence. You may for instance have a stretch which  is T-X-A-X-X-Long, where X is, for example, anything other than A, G, T  or P and Long is anything with a long aliphatic side-chain. If this  motif only appears once in your sequence, then you can make a sequence  specific assignment. If you like, you can immediately enter this into  Analysis. But I tend to wait until I have assigned most of the sequence  and am feeling fairly confident with my assignment before doing this, as  it makes undoing mistakes easier - and when doing double resonance  backbone assignment it is easier to make mistakes than when you are  doing triple resonance assignment! To enter your sequence specific  assignment in Analysis, simply bring up the [B]Assignment Panel[/B] for one of the peaks in one of the spin systems you want to assign. Select a resonance and click [B]Assign {SpinSystem}ATOM[Resonance][/B]. The [B]Atom Browser[/B] panel will appear and you now need to select the exact atom which this resonance corresponds to.
 [IMG]http://www.protein-nmr.org.uk/pictures/assign_practice/seqspec_assignment_text.png[/IMG]
  This atom will then be shaded in a slightly darker colour in the [B]Atom Browser[/B] panel to indicate that it is assigned. If you have [B]Set Sequential Links[/B]  between your spin systems, then you should notice that when you assign  one of the spin systems, all the sequentially linked ones will become  assigned (and shaded in the [B]Atom Browser[/B]) at the same time.
  [URL="http://www.protein-nmr.org.uk/assignment_practice_v2.html#top"]Top[/URL]
    Hints and Tips
 The intensity of peaks varies with their mobility. Sections of the  protein in secondary structure will be fairly rigid. They should all  show similar, reasonably strong, peak intensities. Slightly mobile  regions, such as loop regions tend to show reduced peak intensities and  in the worst case scenario are simply not visible at all. Very highly  flexible regions, such as the C-terminus show very high peak  intensities. Peak intensities will tend to go in waves along the  backbone with neighbouring residues tending to have similar peak  intensities.
 The C-terminus can usually be identified fairly easily due to its  strong peak in a very characteristic part of the HSQC spectrum (see [URL="http://www.protein-nmr.org.uk/spectra.html#hsqc_fig"]here[/URL] for a figure).
 Serine peaks are often rather weak.
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