Dear Rasmus,

that was a really helpful tutorial. I followed the path you pointed out and went through the whole comments.
Thank you very much !

Carolin




On Mar 6, 2012, at 7:51 PM, Rasmus Fogh wrote:

> Dear Carolin,
> 
> It looks to me that the Quality Report does reflect problems with your spectra and assignment - which is indeed what we made it for. Everybody always gets assignment problems, and they need to be found and fixed. At some point people should go through all the errors in the quality reports and either fix them or know that they are OK and why. You are not the first to find that the error messages are not particularly obvious to interpret, though. So, can I use your case to describe how I recommend working?
> 
> The [Help] button will give you the popup help with a general explanation of what things mean, but a step-by-step description is likely more useful here. So here goes:
> 
> First, Errors like 'Impossible bond' or 'excessive bonds' mean that the assignments are not compatible with normal chemical structure - as in pentavalent carbons or N-Cb direct bonds. The most common reasons are wrong experiment types, wrong dimension mappings, or misassignments.
> 
> The best place to start is in M:Assignment:Quality Reports {Resonances}
> 
> Set 'Show only alerts' on (top right). The 'Bound' column shows which atoms are bound to the atom in question. Taking '21AlaN' as a nice example, you see that it is directly bound to six different atoms in residues 20 and 21. Select the row, and press [Show Peaks]. This gives you a popup with all peaks assigned to this resonance. Now look for the peak that says that A21N is directly bound to N20CB. The easiest way to find an assignment is to click on the Assign F1 or Assign F2 column to sort by assignment. You will find that N20CB and N20Cg only appear once, for peaks in NCOCA_32. So the program must think that F1 and F3 in NCOCA_32 are directly bound. Going to M:Experiment:Spectra{Referencing} and selecting NCOCA_32, you can see from the spectral widths that Dim 3 is N, Dim 2 is CO, and dim 1 is CX (as they should be). Go to M:Experiment:Experiments{Experiment types}, and select NCOCA_32. The Type Synonym and Full Type columns look OK. But the table at the bottom left says that Dim 1 (CX) is directly bound to Dim 3 (N) - which is clearly wrong. These mappings can sometimes get set wrongly. To fix it, double click on 1(13C) in line with the onebond transfer, and change it to 2(13C).
> 
> Now go back to the assignment quality report, and click [Update table} Most of the red colour disappears, and most of the red error color in the NCA and NCOCA peak lists disappears as well.
> 
> Next select S7N or S10N, click [Show Peaks], and look for peaks that can cause the erroneous bonds (like S7N-S10CA or S10N S10CB). It looks to me that here the spectrum mappings are right, but that the assignments are wrong in this individual case. You would have to check yourself.
> 
> When you have checked all the error reports in the 'Bound' column, there will still be others. 'repeat' errors in the 'Resonance' column show that the same atom has been used more than once. If all the frequencies fit, you can go to the M:Resonance:Resonances popup, sort the resonances by Residue, select the resonances with the same assignment, and click [Merge]
> 
> Red colour in the SD and 'Max Peak Delta' columns show assignments that are at very different frequencies for the same resonance. Try sorting by 'Max Peak delta', selecting the resonance with the largest delta, and doing [Show peaks]. You can then see which peaks have which frequencies, and check the peak if it looks like it is correclty picked and assigned. Or if the some of trhe psectra need a different referencing or have different shifts caused by different pH or temperature.
> 
> ---------------------------------------------------------------------------
> Dr. Rasmus H. Fogh                  Email: [log in to unmask]
> Dept. of Biochemistry, University of Cambridge,
> 80 Tennis Court Road, Cambridge CB2 1GA, UK.     FAX (01223)766002
> 
> On Tue, 6 Mar 2012, Seuring Carolin wrote:
> 
>> Dear all,
>> 
>> Looking at the Quality Reports for the protein I work on using solid-state NMR data, I get the following assignment error messages for some residues :
>> "Impossible bond" for a residue as ThrCa-ThrN in a NCA or "excessive bonds" for a LysC-LysCa-LysN assignment in a NCaCx.
>> I am very confident that the Quality report is not correct, so I am wondering if this error appears because of one of the following reasons or elsewise.
>> - assumptions CCPN makes, that are only valid in liquid state or
>> - tight set reference rc shifts
>> - or experiment types (I chose N_CA.onebond (through-space) for the NCA and N_CA_C.onebond,through-space for the NCacx)
>> 
>> 
>> 
>> Thank you for your answer!
>> Carolin