Met methyl assignment with NOESY: Difference between revisions

Latest revision as of 16:07, 18 December 2009

The Met methyl group has unique chemical shifts with 1H around 2.0ppm and 13C at 17ppm. Therefore, the best way to identify Met methyl is starting from the aliphatic [13C, 1H]-HSQC. As most proteins have very few methionines, the assignment strategy described below takes a "manual" approach. It is also recommended to obtain aliphatic side-chain assignment before pursuing the following approach.

Open two instances of XEASY: one with the aliphatic [13C, 1H]-HSQC and the other with the simultaneous [1H, 1H]-NOESY. Prepare the NOESY by loading the sequence file, atom list, and peak list. Select only the methionine residues (sp and type MET in the box labeled fragment type and type 0 in the box labeled sequential range) and create the strips.
Go to the HSQC and look for the methionine methyl groups. They should be isolated from most other peaks (around 2ppm in 1H and 17ppm in 13C) and their sign will be opposite of the other methyl peaks in a 28ms constant time HSQC. Note the chemical shifts for each peak.
Go back to the NOESY and look at all of the strips for residue "X". Place the cursor in the last strip of residue "X" and type zd (zoom duplicate) to create another strip. Place the cursor in the new strip and type gp (go to plane). In the pop-up dialog window, type the 13C chemical shift of one of the methyl groups. (NOTE: it is important to type the chemical shift in as "17.65p" to indicate ppm. If you type only "17.65", then you will go to plane 17.) Next, use gr and gl to move the center of the strip to the correct 1H chemical shift.
Compare the peak patterns to determine if the current Met methyl peak corresponds to residue "X". If it does, pick the peak (pp) and assign it (ap). If it does not, go back to step 3 and repeat using the chemical shifts of a different Met methyl from the HSQC.

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-== '''Met Methyl Resonance Assignment''' ==
+The Met methyl group has unique chemical shifts with 1H around 2.0ppm and 13C at 17ppm. Therefore, the best way to identify Met methyl is starting from the aliphatic [13C, 1H]-HSQC. As most proteins have very few methionines, the assignment strategy described below takes a "manual" approach. It is also recommended to obtain aliphatic side-chain assignment before pursuing the following approach.&nbsp;
-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.
-# 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.
+#Open two instances of XEASY:&nbsp;one with the aliphatic [13C, 1H]-HSQC and the other with the simultaneous [1H, 1H]-NOESY. Prepare the NOESY by loading the sequence file, atom list, and peak list. Select only the methionine residues (<tt>sp</tt> and type <tt>MET</tt> in the box labeled <tt>fragment type</tt> and type <span style="font-family: monospace;">0</span><tt></tt> in the box labeled <tt>sequential range</tt>) and create the strips.<br>
+#Go to the HSQC and look for the methionine methyl groups. They should be isolated from most other peaks (around 2ppm in 1H and 17ppm in 13C) and their sign will be opposite of the other methyl peaks in a 28ms constant time HSQC. Note the chemical shifts for each peak.
--- Main.GaohuaLiu - 30 Jan 2008
+#Go back to the NOESY and look at all of the strips for residue "X". Place the cursor in the last strip of residue "X" and type <tt>zd</tt> (zoom duplicate) to create another strip. Place the cursor in the new strip and type <tt>gp</tt> (go to plane). In the pop-up dialog window, type the 13C chemical shift of one of the methyl groups. (NOTE: it is important to type the chemical shift in as "17.65p" to indicate ppm. If you type only "17.65", then you will go to plane 17.) Next, use <tt>gr</tt> and <tt>gl</tt> to move the center of the strip to the correct 1H chemical shift.
+#Compare the peak patterns to determine if the current Met methyl peak corresponds to residue "X". If it does, pick the peak (<tt>pp</tt>) and assign it (<tt>ap</tt>). If it does not, go back to step 3 and repeat using the chemical shifts of a different Met methyl from the HSQC.

Met methyl assignment with NOESY: Difference between revisions

Latest revision as of 16:07, 18 December 2009

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