View source for HA and HB Assignment with GFT in XEASY

== '''HA And HB Assignments with XEASY/UBNMR'''  ==

HA and HB assignments provide the bridge from [[XEASY Backbone Assignment|Backbone Assignment]] to the complete [[XEASY Side Chain Assignments|Side Chain Assignments]]. 

=== '''Analysis of the(4,3)D GFT HABCAB(CO)NHN'''  ===

The sequence-specific 15N / 1HN / 13CAB resonance assignments obtained as described in backbone assignment are first used to also obtain 1HA and 1HB shift assignments by analyzing (4,3)D HABCAB(CO)NHN. Then, shifts of more peripheral aliphatic spins are obtained from (4,3)D HCCH. 

#Go to the directory <tt>/analisys/xeasy/habcab</tt> and copy the <tt>final-clean.seq</tt> and <tt>final-clean.prot</tt> files from the backbone directory 
#Run <tt>makeHabcabPeaks</tt> in [[UBNMR|UBNMR]] to generate an extended [[XEASY Atom List|AtomList]] <tt>habcabconhI1.prot</tt> (containing linear combinations of 13CAB and 1HAB shifts) and the initial HABCAB [[XEASY Peak List|PeakList]] <tt>habcabconhI1.peaks</tt> to guide manual peak identification in (4,3)D HABCAB(CO)NHN. Peaks are colored according to type of sub-spectrum and type of proton involved in the GFT dimension. 
#In XEASY
##use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt> and <tt>lp</tt> to load the sub-spectra of (4,3)D HABCAB(CO)NHN and the starting HABCAB-peak list, then use <tt>se</tt>, <tt>gs</tt> to display [w1(13C,1H),w3(1HN)]-strips and use <tt>mr</tt> to accurately position peaks 
#Use <tt>aa</tt>, <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to update the peak list as <tt>habcabconhO1.peaks</tt> and AtomList as <tt>habcabconhO1.prot</tt>. See the analysis procedure below. 
#In XEASY, use <tt>ns</tt> to load subspectrum I, <tt>HABCABcoNH1</tt>, from <tt>/protName/analysis/xeasy/data/</tt>; <tt>ls</tt> to load the sequence file, e.g. <tt>protein.seq</tt>; <tt>lc</tt> to load AtomList from <tt>habcabconhI1.prot</tt>; <tt>lp</tt> to load the starting peaklist <tt>habcabconhI1.peaks</tt>; use <tt>ns</tt> to load sub-spectrum II, <tt>HABCABcoNH2</tt>.

#In XEASY, use <tt>cp</tt> to display the spectrum as a countour plot; use <tt>fp</tt> or <tt>bp</tt> to step through the planes until you see representative peaks for setting the coutour levels; use <tt>pm</tt> to display [w1(13C/1H),w2(15N)]-planes; use <tt>mr</tt> to accurately position the peaks along w2(15N); use <tt>pm</tt> to display [w2(15N),w3(1HN)]-planes. 
#In XEASY, use <tt>se</tt> and <tt>gs</tt> to create strips from entries of loaded PeakList and display, for example, ten at once; navigate through the strips by using <tt>fs</tt> and <tt>bs</tt>; use <tt>pw</tt> to set the '''assignment y''' in the peak window so that only the assignment in the GFT dimension is displayed. One should see strips from both sub-spectra assigned to the same residue side-by-side. 
#In XEASY, use <tt>gs</tt>, <tt>sf</tt>, <tt>fs</tt> and <tt>bs</tt> to go through strips and use <tt>mr</tt> to move the peaks to the actual peak positions. Make sure that the peaks are moved in the right sub-spectrum. If two HB-proton shifts are apparently degenerate, move the corresponding peak to the same position (note that in the better resolved NOESY, the two shifts may turn out to be non-degenerate). If the assignment is ambiguous, load 'central peak spectrum' to identify peak pairs in the sub-spectra. <br> '''Figure 1: Example of (4,3)D GFT HABCAB(CO)NHN analysis before (left) and after (right) peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY hab5.jpg]] [[Image:XEASY hab4.jpg]] <br> 
#In UBNMR, run <tt>updateHabAtom</tt> to update single-quantum 1HAB and 13CAB shifts as <tt>habcabconhO2.prot</tt>. A least-squares fit provides the single-quantum HA/HB chemical shifts (consistency of peaks representing different linear combinations of shifts is checked by UBNMR in oder to identify inadvertently 'mis-picked' peaks; a warning is then provided). 
#In XEASY, double-check the peak positions for residues that gaving large error. Repeat previous step and this step till no improvement can be made. 
#Now it is ready to move to [[XEASY Side Chain Assignments|Side Chain Assignments]]

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*[[Media:XEASY_makeHabcabPeaks.txt|makeHabcabPeaks]]: UBNMR macro

*[[Media:XEASY_updateHabAtom.txt|updateHabAtom]]: UBNMR macro