HNCACAB/CABCA(CO)NH - Revision history

Jlmills at 21:29, 30 November 2009

2009-11-30T21:29:41Z

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 ##In XEASY, repeat 5 and 6 until analysis is complete
 ##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]] which should be saved, and re-loaded and saved a second time.
-##In UBNMR, run [[Media:cleanBbGftProt.txt|cleanBbGftProt]]cleanBbGftProt to make a clean [[XEASY Atom List|AtomList]] and [[XEASY Sequence List|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
+##In UBNMR, run [[Media:CleanBbGftProt.txt|cleanBbGftProt]] to make a clean [[XEASY Atom List|AtomList]] and [[XEASY Sequence List|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.<br>
 <br>

Jlmills at 21:27, 30 November 2009

2009-11-30T21:27:17Z

← Older revision		Revision as of 21:27, 30 November 2009
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	##In XEASY, repeat 5 and 6 until analysis is complete		##In XEASY, repeat 5 and 6 until analysis is complete
	##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List\|SequenceList]], [[XEASY Atom List\|AtomList]] and [[XEASY Peak List\|PeakList]] which should be saved, and re-loaded and saved a second time.		##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List\|SequenceList]], [[XEASY Atom List\|AtomList]] and [[XEASY Peak List\|PeakList]] which should be saved, and re-loaded and saved a second time.
	##In UBNMR, run cleanBbGftProt to make a clean [[XEASY Atom List\|AtomList]] and [[XEASY Sequence List\|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.		##In UBNMR, run [[Media:cleanBbGftProt.txt\|cleanBbGftProt]]cleanBbGftProt to make a clean [[XEASY Atom List\|AtomList]] and [[XEASY Sequence List\|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
	<blockquote><pre>#UBNMR macro cleanBbGftProt		<blockquote><pre>#UBNMR macro cleanBbGftProt

Jlmills at 21:22, 30 November 2009

2009-11-30T21:22:45Z

Jlmills at 18:06, 30 November 2009

2009-11-30T18:06:20Z

Show changes

Jlmills at 17:01, 30 November 2009

2009-11-30T17:01:40Z

← Older revision		Revision as of 17:01, 30 November 2009
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	== '''Backbone Assignment with XEASY/UBNMR''' ==		== '''Backbone Assignment with XEASY/UBNMR''' ==

	Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments HNNCACB/CACBCONHN.		Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH\|HNNCACB/CACBCONHN]].

	=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===		=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

	HNNCABCA ~~comprises of~~ peaks representing both intra-residue and sequential connectivities (as in HNNCACB)~~. These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure~~. Since the ~~intra-residue~~ connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).		The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure.<br>

	~~<br>~~

	~~#'''Peak Picking''' <br>~~
	##Go to <tt>analysis/xeasy/backbone</tt>~~. Edit~~ macro <tt>getfil</tt> to import backbone spectra, ~~sequence~~, ~~prot~~ and ~~peaklists~~. Edit ~~macro~~ [[Media:XEASY_makeCabcaPeak.txt\|makeCabcaPeak]] ~~with~~ the sequence and ~~prot~~ file names. In UBNMR, run ~~macro~~ <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList ~~for backbone assignment which~~ contains linear combinations of 13CA and 13CB shifts for each residue and SRD~~; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I~~. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. ~~<br>~~		#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY_Sequence_List\|SequenceList]], [[XEASY_Atom_List\|AtomList]] and [[XEASY_Peak_List\|PeakLists]].
	##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ~~ProtonList~~ and PeakList~~; use~~ <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A)~~; use~~ <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)~~; use~~ <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY backbone2.jpg]] <br> ~~<br>~~		#Edit the [[Media:XEASY_makeCabcaPeak.txt\|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY_Atom_List\|AtomList]] and [[XEASY_Peak_List\|PeakList]]. The new [[XEASY_Atom_List\|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
			#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY_Sequence_List\|SequenceList]], [[XEASY_Atom_List\|AtomList]] and [[XEASY_Peak_List\|PeakList]]
			#Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A)
			#Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
			#Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY backbone2.jpg]] <br>

	#'''Initial Backbone Assignment from AutoAssign''' <br>		#'''Initial Backbone Assignment from AutoAssign''' <br>
	##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README.txt\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''		##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README.txt\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''
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	</pre></blockquote>		</pre></blockquote>
	<br>		<br>

	=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

Agutmana at 22:21, 11 November 2009

2009-11-11T22:21:05Z

← Older revision		Revision as of 22:21, 11 November 2009
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	#'''Confirming Backbone Assignment from AutoAssign in XEASY''' <br> Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY '''Also see below the use Of 15N-resolved [1H,1H] NOESY Spectrum).''' <br>		#'''Confirming Backbone Assignment from AutoAssign in XEASY''' <br> Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY '''Also see below the use Of 15N-resolved [1H,1H] NOESY Spectrum).''' <br>
	##In UBNMR, run macro [[~~NESG~~:~~%ATTACHURL%/makeBbNNoesy~~\|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only contains diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.		##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt\|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only contains diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
	##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; <tt>lc</tt> to load the AtomList; use <tt>lp</tt> to load the CABCA-PeakList.		##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; <tt>lc</tt> to load the AtomList; use <tt>lp</tt> to load the CABCA-PeakList.
	##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.		##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.

Agutmana at 22:20, 11 November 2009

2009-11-11T22:20:18Z

Agutmana at 19:37, 10 November 2009

2009-11-10T19:37:29Z

← Older revision		Revision as of 19:37, 10 November 2009
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	<br> Modified macro for AA2XEASY		<br> Modified macro for AA2XEASY
	<blockquote><pre>		<blockquote><pre>#UBNMR script for converting autoassign results to xeasy format sequence
	#UBNMR script for converting autoassign results to xeasy format sequence

	init read seq ../nhsqc.seq		init read seq ../nhsqc.seq
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	##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.		##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.

	~~#UBNMR macro cleanBbGftProt~~		<blockquote><pre>#UBNMR macro cleanBbGftProt
	<blockquote><pre>init
			init

	read seq nhsqc.seq		read seq nhsqc.seq
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	</pre></blockquote>		</pre></blockquote>
	<br>		<br>

	=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

Agutmana at 19:35, 10 November 2009

2009-11-10T19:35:46Z

← Older revision		Revision as of 19:35, 10 November 2009
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	#'''Peak Picking''' <br>		#'''Peak Picking''' <br>
	##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>		##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>
	##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[~~File~~:~~XEASY_backbone2~~.jpg]] <br> <br>		##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY backbone2.jpg]] <br> <br>
	#'''Initial Backbone Assignment from AutoAssign''' <br>		#'''Initial Backbone Assignment from AutoAssign''' <br>
	##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''		##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''
	##***Click for the [[AutoAssign]] control file example [[Media:XEASY_myprot.aat\|myprot.aat]].		##:*Click for the [[AutoAssign]] control file example [[Media:XEASY_myprot.aat\|myprot.aat]].
	##***Peak pattern used in the [[AutoAssign]] input file of 3D CACB type experiments: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;		##:*Peak pattern used in the [[AutoAssign]] input file of 3D CACB type experiments: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
	##***Peak pattern used in the [[AutoAssign]] input file of 4D CACB type experiments: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt>; and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>.<br>		##:*Peak pattern used in the [[AutoAssign]] input file of 4D CACB type experiments: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt>; and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>.<br>
	##Run [[AutoAssign]] several times with varying matching tolerances for CA and CB in the myprot.aat file (0.1-0.6ppm); save AutoAssign output files;<br>		##Run [[AutoAssign]] several times with varying matching tolerances for CA and CB in the myprot.aat file (0.1-0.6ppm); save AutoAssign output files;<br>
	##***It is suggested to use <tt>Default Execution</tt> method to run the program.		##:*It is suggested to use <tt>Default Execution</tt> method to run the program.
	##***From the main menu of [[AutoAssign]], follow option <tt>Examine</tt> > <tt>All GSs</tt> to write output file that contains both AutoAssign assignment and the corresponding '''SRD-I''' residue numbers. <br>		##:*From the main menu of [[AutoAssign]], follow option <tt>Examine</tt> > <tt>All GSs</tt> to write output file that contains both AutoAssign assignment and the corresponding '''SRD-I''' residue numbers. <br>
	##In UBNMR, run macro [[Media:AA2Xeasy\|AA2Xeasy]] to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with <tt>mapping numbers</tt>. <br> <br>		##In UBNMR, run macro [[Media:AA2Xeasy\|AA2Xeasy]] to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with <tt>mapping numbers</tt>. <br> <br>

	<br> Modified macro for AA2XEASY		<br> Modified macro for AA2XEASY
	<blockquote><pre>#UBNMR script for converting autoassign results to xeasy format sequence		<blockquote><pre>
			#UBNMR script for converting autoassign results to xeasy format sequence

	init read seq ../nhsqc.seq		init read seq ../nhsqc.seq

	#update autoassign results to xeasy sequence		#update autoassign results to xeasy sequence

	update mapping aa.out 200		update mapping aa.out 200

	#200 is the number difference between self and sequencial SRD's.		#200 is the number difference between self and sequencial SRD's.

	write updated sequence		write updated sequence

	write sequence aa.seq		write sequence aa.seq
	</pre></blockquote>		</pre></blockquote>
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	</pre></blockquote>		</pre></blockquote>
	<br>		<br>

	=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

Agutmana at 19:33, 10 November 2009

2009-11-10T19:33:50Z

← Older revision		Revision as of 19:33, 10 November 2009
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	#'''Peak Picking''' <br>		#'''Peak Picking''' <br>
	##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>		##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>
	##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[~~File~~:~~XEASY_backbone1~~.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[~~Image~~:~~Backbone2~~.jpg]] <br> <br>		##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[File:XEASY_backbone2.jpg]] <br> <br>
	#'''Initial Backbone Assignment from AutoAssign''' <br>		#'''Initial Backbone Assignment from AutoAssign''' <br>
	##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''		##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''

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	#'''Initial Backbone Assignment from AutoAssign''' <br>		#'''Initial Backbone Assignment from AutoAssign''' <br>
	##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt\|makeAutoList]] and file <tt>myprot.aat</tt>.		##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt\|makeAutoList]] and file <tt>myprot.aat</tt>.
	##Run <tt>makeAutoList</tt> in [[UBNMR\|UBNMR]] to generate input files for [[AutoAssign\|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''		##Run <tt>makeAutoList</tt> in [[UBNMR\|UBNMR]] to generate input files for [[AutoAssign\|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID of the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''
	###Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;		##Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
	###Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>.<br>		##Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>.<br>
	###Run [[AutoAssign]] several times with varying matching tolerances for CA and CB in the myprot.aat file ~~(0.1-0.6ppm);~~ save [[AutoAssign\|AutoAssign]] output files<br>		##Run [[AutoAssign]] several times (using <tt>Default Execution</tt>) with varying matching tolerances (0.1-0.6ppm) for CA and CB in the <tt>myprot.aat</tt> file and save [[AutoAssign\|AutoAssign]] output files<br>
	~~###It is suggested to use <tt>Default Execution</tt> method to run the program.~~		##From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign\|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. <br>
	###From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign\|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. <br>		##In [[UBNMR\|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt\|AA2Xeasy]] to use the best or consensus [[AutoAssign\|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY\|XEASY]] SequenceList with <tt>mapping numbers</tt>.
	###In [[UBNMR\|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt\|AA2Xeasy]] to use the best or consensus [[AutoAssign\|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY\|XEASY]] SequenceList with <tt>mapping numbers</tt>.		#'''Confirming Backbone Assignment from AutoAssign in XEASY''' <br> Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
	#'''Confirming Backbone Assignment from AutoAssign in XEASY''' <br> Here two XEASY sessions are recommended for efficiency, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY		##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt\|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that contains only diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
	##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt\|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only ~~contains~~ diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.		##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the [[XEASY Sequence List\|SequenceList]] that contains [[AutoAssign\|AutoAssign]] results; <tt>lc</tt> to load the [[XEASY Atom List\|AtomList]]; use <tt>lp</tt> to load the CABCA-[[XEASY Peak List\|PeakList]].
			##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the [[XEASY Sequence List\|SequenceList]] that contains [[AutoAssign\|AutoAssign]] results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
	##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; <tt>lc</tt> to load the AtomList; use <tt>lp</tt> to load the CABCA-PeakList.		##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the [[XEASY Sequence List\|SequenceList]] (with [[AutoAssign\|AutoAssign]] assignment) and the [[XEASY Atom List\|AtomList]]; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues.
	##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
	##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the SequenceList (with AutoAssign assignment) and the ~~atomlist~~; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues ~~in SRD -1 sequential order~~.
	##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.		##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
	##Continue to complete the backbone assignment manually as described below		##Continue to complete the backbone assignment manually as described below
	#'''~~Complete~~ Backbone Assignment ~~by perform~~ sequential ordering of SRDs''' <br> Here two XEASY sessions are recommended for efficiency ~~reasons,~~ one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY.		#'''Completing Backbone Assignment using sequential ordering of SRDs''' <br> Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
	##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".		##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
	##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.		##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
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	##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.		##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
	##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.		##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
	##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the SRD-I and SRD-II numbers to residue numbers.		##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the '''SRD-I''' and '''SRD-II''' numbers to residue numbers.
	##In XEASY, repeat 5 and 6 until analysis is complete		##In XEASY, repeat 5 and 6 until analysis is complete
	##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.		##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List\|SequenceList]], [[XEASY Atom List\|AtomList]] and [[XEASY Peak List\|PeakList]] which should be saved, and re-loaded and saved a second time.
	##In UBNMR, run ~~<tt>~~cleanBbGftProt~~</tt>~~ to make a clean AtomList and ~~SeqList~~ by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.		##In UBNMR, run cleanBbGftProt to make a clean [[XEASY Atom List\|AtomList]] and [[XEASY Sequence List\|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
	<blockquote><pre>#UBNMR macro cleanBbGftProt		<blockquote><pre>#UBNMR macro cleanBbGftProt

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	#'''Peak Picking''' <br>		#'''Peak Picking''' <br>
	##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>		##Go to <tt>analysis/xeasy/backbone</tt>. Edit macro <tt>getfil</tt> to import backbone spectra, sequence, prot and peaklists. Edit macro [[Media:XEASY_makeCabcaPeak.txt\|makeCabcaPeak]] with the sequence and prot file names. In UBNMR, run macro <tt>makeCabcaPeak</tt> to generate an extended GFT AtomList for backbone assignment which contains linear combinations of 13CA and 13CB shifts for each residue and SRD; to generate a starting peaklist for analysis of HNNCABCA and CABCA(CO)NHN by using the 15N/1HN backbone shifts of SRD-I. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process. <br>
	##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY backbone2.jpg]] <br> <br>		##In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the SequenceList, ProtonList and PeakList; use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A); use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks); use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. <br> <br> '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' <br> '''A: Before peak position adjustment by <tt>mr</tt>;''' <br> [[Image:XEASY backbone1.jpg]]<br> <br> '''B: After peak position adjustment by <tt>mr</tt>.''' <br> [[Image:XEASY backbone2.jpg]] <br> <br>
	#'''Initial Backbone Assignment from AutoAssign''' <br>		#'''Initial Backbone Assignment from AutoAssign''' <br>
	##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''		##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README.txt\|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt\|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. <br> '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''
	##:*Click for the [[AutoAssign]] control file example [[Media:XEASY_myprot.aat\|myprot.aat]].		##:*Click for the [[AutoAssign]] control file example [[Media:XEASY_myprot.aat\|myprot.aat]].
	##:*Peak pattern used in the [[AutoAssign]] input file of 3D CACB type experiments: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;		##:*Peak pattern used in the [[AutoAssign]] input file of 3D CACB type experiments: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
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	##:*It is suggested to use <tt>Default Execution</tt> method to run the program.		##:*It is suggested to use <tt>Default Execution</tt> method to run the program.
	##:*From the main menu of [[AutoAssign]], follow option <tt>Examine</tt> > <tt>All GSs</tt> to write output file that contains both AutoAssign assignment and the corresponding '''SRD-I''' residue numbers. <br>		##:*From the main menu of [[AutoAssign]], follow option <tt>Examine</tt> > <tt>All GSs</tt> to write output file that contains both AutoAssign assignment and the corresponding '''SRD-I''' residue numbers. <br>
	##In UBNMR, run macro [[Media:~~AA2Xeasy~~\|AA2Xeasy]] to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with <tt>mapping numbers</tt>. <br> <br>		##In UBNMR, run macro [[Media:XEASY_AA2Xeasy.txt\|AA2Xeasy]] to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with <tt>mapping numbers</tt>. <br> <br>

	<br> Modified macro for AA2XEASY		<br> Modified macro for AA2XEASY
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	##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.		##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.
	##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.		##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.

	<blockquote><pre>#UBNMR macro cleanBbGftProt		<blockquote><pre>#UBNMR macro cleanBbGftProt