Amide Side Chain assignment with NOESY: Difference between revisions

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== '''Amide Side-chain Resonance Assignment Using The Simultaneous 15N, 13C-resolved (1H,1H) NOESY''' ==
== '''Amide Side chain resonance assignment using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY''' ==


The amide side-chain resonance assignment here means the assignment of amide side-chain of residues Asn and Gln, which can be done while doing the aliphatic side-chain assignment if the NOESY is used. The approach described below focuses on the simultaneous 15N, 13C-resolved (1H,1H) NOESY, please refer to [[NESG:Side Chain Assignments Using Simultaneous 15N,13C-resolved(1H,1H)NOESY|Side Chain Assignments Using Simultaneous 15N,13C-resolved(1H,1H)NOESY]] for addtional information. This approach can also be applied to a combined use of 15N-(1H,1H) NOESY and 13C-resolved (1H,1H) NOESY. <br/> ''NOTE:  It is recommended to obtain aliphatic side-chain assignment before pursuing the following approach.''
This section describes the procedure to assign the amide side chains of Asn (d2) and Gln (e2) using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY (or by combining separate 15N resolved and 13C-resolved [1H, 1H]-NOESY spectra). Assuming that you have complete assignments of the Asn/Gln aliphatic side chain, extending the assignment to include the d2 and e2 is straightforward.  
# Process the simultaneous NOESY as a single 13C NOESY spectra. In this case, the 15N chemical shift is calibrated as 13C shift. The real chemical shift can be easily calculate back based on the aquisition parameters. It is important to analyse the simultaneous NOESY as a real single NOESY.
# In UBNMR, run <tt>makeNoePeaks</tt> to generate  a starting simultaneous NOESY PeakList  including intra- and sequential NOEs, considering previously assigned chemical shifts and also averaged chemical shifts from BMRB for all assignable unassigned amide side-chain resonances. The 15N chemical shift has already been converted to pseudo-13C chemical shift which will exacltly fit the spectra.
# In XEASY, use <tt>ns</tt> to load  two copies of the Simultaneous 15N,13C-resolved (1H,1H) NOESY and display the two copies in different views (X-axis :w1(13C/15N), Y-axis: w2(1H), Z-axis: w3(1HC/HN) & X-axis: w3(1HC/HN), Y-axis: w2(1H), Z-axis: w1(13C/15N)  use <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt>, respectively, to load noe.seq, bbsc.prot and simnoesyI1.peaks; use <tt>sp</tt> to select peaks of residues Asn; use <tt>se</tt> and <tt>gs</tt> to display [w2(1H),w3(1H)]-strips and  [w2(1H),w1(13C)]-strips  residue by residue.
# In XEASY, go to the CB-strips of each Asn residue. Two strong peaks corresponding to the HB-HD21 and HB-HD22 NOEs are expected at around 7 ppm along w2(1H, indirect dimension); use <tt>mr</tt> to move peaks to the identified peaks. Move to the most possible (strongest) two if there are many candidates.
# In XEASY, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to resort and re-display the [w2(1H),w3(1H)]-strips and  [w2(1H),w1(13C)]-strips  for residues Asn. Since the chemical shift of HD21 & HD22 are very likely correct, identify the correct ND2 chemical shift in the corresponding ND2/HD21 and ND2/HD22  strips with view (X-axis: w3(15N), Y-axis:w2(1H); z: w3(1H)) based on the expected peak pattern and expected ND2 chemical shifts. Use <tt>zd</tt> to duplicate the ND2 strips if the expect position of ND2 is out of the strip view. Use <tt>gl</tt> or <tt>gr</tt> to change the dulplicate strip view to find the expected ND2; use <tt>mr</tt> to move the peak positions. Please also see [[NESG:Aliphatic Side-chain Resonance Assignment Using The Aliphatic HCCHCOSY Spectrum|Aliphatic Side-chain Resonance Assignment Using The Aliphatic HCCHCOSY Spectrum]] for addition information.  Go back to the the CB-strips and identify the possible HD21/HD22 if no expected peak pattern cound be found for the initially identified HD21/HD22.
# In XEASY, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to resort and re-display the [w2(1H),w3(1H)]-strips and  [w2(1H),w1(13C)]-strips, double check the assignment obtained.
# In XEASY, assign the amide side-chain of '''Gln''' residues in the same manner. Go to the CG-strips of each Gln residue indead of CB-strips for Asn residue. Strong peaks corresponding to the HG-HE21 and HG-HE22 NOEs are expected at around 7 ppm along w2(1H); use <tt>mr</tt> to move peaks to the identified peaks. Assign the Gln sidechain NE2 and the HE21 and HE22 peaks as described for Asp.
# In XEASY, use <tt>wp</tt> to write an updated peaklist as noesyNO1.peaks.  


<br>


== '''Met Methyl Resonance Assignment''' ==
#Load two copies of the NOESY into XEASY. Be sure to use two different orientations for the two spectra. Spectrum I should be viewed as 13C/15N(x-axis), indirect 1H(y-axis), and direct 1H(z-axis) while spectrum II&nbsp;should be viewed as direct 1H(x-axis), indirect 1H(y-axis), and 13C/15N (z-axis). Additionally, load the appropriate peak list, sequence list, and atom list. Select Asn and Gln peaks with <tt>sp</tt> (type <tt>ASN GLN</tt> in the box labeled <tt>fragment type</tt> and type <tt>0</tt> in the box labeled <tt>sequential range</tt>). Create strips in both spectra (<tt>es</tt>, <tt>se</tt>, <tt>rc</tt>, <tt>rs</tt>, <tt>se</tt>, <tt>gs</tt>).
#Look at the CB strips of Asn or the CG strips of Gln. You should see 2 strong cross peaks near 7ppm in both the HB2(HG2) and HB3(HG3) strips which correspond to HB-HD21/HB-HD22 or HG-HE21/HG-HE22. At this point, it is helpful to create 4 new peaks (ND2/HD22/HD22, ND2, HD21/HD21, ND2/HD22/HD21, and ND2/HD21/HD22 for Asn and NE2/HE22/HE22, NE2, HE21/HE21, NE2/HE22/HE21, and NE2/HE21/HE22 for Gln. The location of these peaks is not important at this time because they will be moved to the proper location.) Once you have identified the 2 amide side chain peaks and created the new peaks, use <tt>mr</tt> to move the HD22/HD21 (or HE22/HE21) peaks in spectrum II (the one with the direct 1H dimension along the x-axis).<br>
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to see the updated peak positions and the newly created peaks. Use <tt>mr</tt> again to move the peaks in spectrum I.<br>
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to verify the new assignments.<br>


Usually, the Met methyl resonance assignment can be obtained from NOESY, however, in a different manner as described above. The Met methyl group has unique chemical shifts with 1H around 2.0ppm and 13C as 17ppm. Therefore, the best way to identify Met methyl is starting from the 13C aliphatic HSQC. It is also recommended to obtain aliphatic side-chain assignment before pursuing the following approach.
<br> -- Main.GaohuaLiu - 25 Mar 2007
# Same as above, process the simultaneous NOESY, make a 2D 13C HSQC project plane for the NOESY, generate  a starting simultaneous NOESY PeakList from a AtomList with Met methy chemical shifts.
# In XEASY, load the 3D NOESY PeakList to the 2D 13C aliphatic HSQC or the 2D 13C HSQC projection plane, type in the dimention names as below in the pop-up window. <br/>       <img src="%ATTACHURLPATH%/3d2d.jpg" alt="3d2d.jpg" width='389' height='147' /> <br/> use <tt>pp</tt> to manually pick the possible Met methyl peaks in the 2D spectrum, use <tt>wr</tt> write the update NOESY PeakList.
# In XEASY, load the two copies of 3D NOESY and the updated 3D PeakList; use <tt>sp</tt>, <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to create and display strips from Met residues in two orthogonal views; use <tt>sp</tt>, <tt>se</tt> to create addtional strips for these manually unassigned peaks. ''Note: All the other peaks from the NOESY are simulated and assigned; type in <tt>n= and =n</tt> when the window below pop up.'' <br/>      <img src="%ATTACHURLPATH%/strip1.jpg" alt="strip1.jpg" width='388' height='120' />  <br/>
# In XEASY, use <tt>cd</tt>, <tt>cc</tt> to identify these unassigned peaks to the corresponding Met methyl group, use <tt>mr</tt> to adjust the simulated Met methyl peaks to the assign position, use <tt>dp</tt> to delete the manually picked peaks  that are aready indentified.
# In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated AtomList and PeakList.
 
 
-- Main.GaohuaLiu - 25 Mar 2007

Revision as of 23:40, 10 December 2009

Amide Side chain resonance assignment using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY

This section describes the procedure to assign the amide side chains of Asn (d2) and Gln (e2) using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY (or by combining separate 15N resolved and 13C-resolved [1H, 1H]-NOESY spectra). Assuming that you have complete assignments of the Asn/Gln aliphatic side chain, extending the assignment to include the d2 and e2 is straightforward.


  1. Load two copies of the NOESY into XEASY. Be sure to use two different orientations for the two spectra. Spectrum I should be viewed as 13C/15N(x-axis), indirect 1H(y-axis), and direct 1H(z-axis) while spectrum II should be viewed as direct 1H(x-axis), indirect 1H(y-axis), and 13C/15N (z-axis). Additionally, load the appropriate peak list, sequence list, and atom list. Select Asn and Gln peaks with sp (type ASN GLN in the box labeled fragment type and type 0 in the box labeled sequential range). Create strips in both spectra (es, se, rc, rs, se, gs).
  2. Look at the CB strips of Asn or the CG strips of Gln. You should see 2 strong cross peaks near 7ppm in both the HB2(HG2) and HB3(HG3) strips which correspond to HB-HD21/HB-HD22 or HG-HE21/HG-HE22. At this point, it is helpful to create 4 new peaks (ND2/HD22/HD22, ND2, HD21/HD21, ND2/HD22/HD21, and ND2/HD21/HD22 for Asn and NE2/HE22/HE22, NE2, HE21/HE21, NE2/HE22/HE21, and NE2/HE21/HE22 for Gln. The location of these peaks is not important at this time because they will be moved to the proper location.) Once you have identified the 2 amide side chain peaks and created the new peaks, use mr to move the HD22/HD21 (or HE22/HE21) peaks in spectrum II (the one with the direct 1H dimension along the x-axis).
  3. Recreate the strips (es, se, se, gs) to see the updated peak positions and the newly created peaks. Use mr again to move the peaks in spectrum I.
  4. Recreate the strips (es, se, se, gs) to verify the new assignments.


-- Main.GaohuaLiu - 25 Mar 2007

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