Aromatic side chain assignment with Aro-HCCH-COSY in XEASY: Difference between revisions
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Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[NESG:Aliphatic side-chain resonance assignment using the aliphatic HCCH COSY spectrum|aliphatic (4,3)D HCCH Analysis]] for comparison. | |||
Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm. | |||
'''Phe, Tyr, and His''' | |||
*Run the following macro in UBNMR to generate a peaklist and atom list:<br> | |||
<pre> init | |||
read seq myprot.seq #change filename to something appropriate | |||
read prot noe.prot #change filename to your most current prot list | |||
add GFTatom COSY_C ATTACHED_H | |||
simulate 3D CG COSY_H 0 1 | |||
simulate 3D CG COSY_C 0 1 | |||
simulate 3D CG1 COSY_CH 0 1 | |||
simulate 3D CG1 COSY_C 0 1 | |||
simulate 3D CG2 COSY_CH 0 1 | |||
simulate 3D CG2 COSY_C 0 1 | |||
simulate 3D CD COSY_CH 0 2 | |||
simulate 3D CD COSY_C 0 2 | |||
simulate 3D CD1 COSY_CH 0 2 | |||
simulate 3D CD1 COSY_C 0 2 | |||
simulate 3D CD2 COSY_CH 0 2 | |||
simulate 3D CD2 COSY_C 0 2 | |||
simulate 3D CZ2 COSY_CH 0 3 | |||
simulate 3D CZ2 COSY_C 0 3 | |||
simulate 3D CZ3 COSY_CH 0 3 | |||
simulate 3D CZ3 COSY_C 0 3 | |||
simulate 3D CE3 COSY_CH 0 4 | |||
simulate 3D CE3 COSY_C 0 4 | |||
simulate 3D CH2 COSY_CH 0 4 | |||
simulate 3D CH2 COSY_C 0 4 | |||
write prot hcchAroI1.prot #the newly created starting prot list | |||
write peaks hcchAroI1.peaks #the newly created starting peak list | |||
</pre> | |||
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips. In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts; use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList. In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat 1.- 3. and 4. for <tt>QE strips</tt> and assign HE. Note that for His, assignment is often complicated by the presence of strong signals from HIS-Tags introduced to facilitate protein purification. The QE strip in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possible 13CZ±1HZ (for Phe only). .See also: [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]]. | |||
*'''TRP''' | |||
##In XEASY, for residue Trp, repeat steps 1.-3. for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3. See also [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]]. | |||
##In XEASY, use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save the final PeakList and AtomList | |||
## | |||
## In XEASY, use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save the final | |||
-- Main.GaohuaLiu - 01 Feb 2007 | -- Main.GaohuaLiu - 01 Feb 2007 |
Revision as of 16:37, 18 December 2009
Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See aliphatic (4,3)D HCCH Analysis for comparison.
Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.
Phe, Tyr, and His
- Run the following macro in UBNMR to generate a peaklist and atom list:
init read seq myprot.seq #change filename to something appropriate read prot noe.prot #change filename to your most current prot list add GFTatom COSY_C ATTACHED_H simulate 3D CG COSY_H 0 1 simulate 3D CG COSY_C 0 1 simulate 3D CG1 COSY_CH 0 1 simulate 3D CG1 COSY_C 0 1 simulate 3D CG2 COSY_CH 0 1 simulate 3D CG2 COSY_C 0 1 simulate 3D CD COSY_CH 0 2 simulate 3D CD COSY_C 0 2 simulate 3D CD1 COSY_CH 0 2 simulate 3D CD1 COSY_C 0 2 simulate 3D CD2 COSY_CH 0 2 simulate 3D CD2 COSY_C 0 2 simulate 3D CZ2 COSY_CH 0 3 simulate 3D CZ2 COSY_C 0 3 simulate 3D CZ3 COSY_CH 0 3 simulate 3D CZ3 COSY_C 0 3 simulate 3D CE3 COSY_CH 0 4 simulate 3D CE3 COSY_C 0 4 simulate 3D CH2 COSY_CH 0 4 simulate 3D CH2 COSY_C 0 4 write prot hcchAroI1.prot #the newly created starting prot list write peaks hcchAroI1.peaks #the newly created starting peak list
- In XEASY, use ns to load the three sub-spectra of aromatic (4,3)D HCCH; use ls, lp and lc to load the sequence, peak list, and chemical shift list, respectively and se, gs to sort and display [w1(13C;1H);w3(1H)]-strips. In XEASY, use pm, es, se and gs (or sf) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use mr to accurately adjust peak positions to assign 13CD chemical shifts; use pm and gs to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use mr to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use mr to accurately position peaks along w1; use ac, wp and wc to save updated PeakList and AtomList. In UBNMR, run updatacosyGFT to calculate SQ shifts from updated AtomList. Repeat 1.- 3. and 4. for QE strips and assign HE. Note that for His, assignment is often complicated by the presence of strong signals from HIS-Tags introduced to facilitate protein purification. The QE strip in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possible 13CZ±1HZ (for Phe only). .See also: Assign Aromatic Side-chain Resonances using the NOESY and HCCH.
- TRP
- In XEASY, for residue Trp, repeat steps 1.-3. for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3. See also Assign Aromatic Side-chain Resonances using the NOESY and HCCH.
- In XEASY, use ac, wp and wc to save the final PeakList and AtomList
-- Main.GaohuaLiu - 01 Feb 2007