XEASY Spin system identification - Revision history

Jlmills at 18:33, 24 November 2009

2009-11-24T18:33:26Z

← Older revision		Revision as of 18:33, 24 November 2009
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	==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====		==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

	#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta\|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR\|UBNMR]] to generate an [[XEASY\|XEASY]] [[XEASY Sequence List\|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List\|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY\|XEASY]] Files for book-keeping: [[~~XEASY_Atom_List~~\|AtomList]], [[~~XEASY_Sequence_List~~\|SequenceList]], and [[~~XEASY_Peak_List~~\|PeakList]].		#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta\|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR\|UBNMR]] to generate an [[XEASY\|XEASY]] [[XEASY Sequence List\|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List\|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY\|XEASY]] Files for book-keeping: [[XEASY Atom List\|AtomList]], [[XEASY Sequence List\|SequenceList]], and [[XEASY Peak List\|PeakList]].
	#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY\|XEASY]] library and [[~~XEASY_Sequence_List~~\|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[~~XEASY_Atom_List~~\|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[~~XEASY_Peak_List~~\|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[~~XEASY_Atom_List~~\|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[~~XEASY_Atom_List~~\|AtomList]] entries corresponding to '''SRD-I'''.		#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY\|XEASY]] library and [[XEASY Sequence List\|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY Atom List\|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY Peak List\|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY Atom List\|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY Atom List\|AtomList]] entries corresponding to '''SRD-I'''.

	<br><br>'''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum		<br><br>'''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

	'''A: After in-phase peak picking'''<br> [[Image:XEASY hsqc2.jpg]] <br>		'''A: After in-phase peak picking'''<br> [[Image:XEASY hsqc2.jpg]] <br> <br>
	<br>

	'''B: XEASY <tt>ar</tt> window'''<br> [[Image:XEASY hsqc ar.jpg]]<br>		'''B: XEASY <tt>ar</tt> window'''<br> [[Image:XEASY hsqc ar.jpg]]<br>
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	[[Image:XEASY hsqc3.jpg]]<br>		[[Image:XEASY hsqc3.jpg]]<br>

			<br>

	==== '''Analysis of the 3D HNNCO Spectrum''' ====		==== '''Analysis of the 3D HNNCO Spectrum''' ====
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	#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (<span style="font-family: monospace;">a</span><tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).		#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (<span style="font-family: monospace;">a</span><tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
	#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY Atom List\|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY Peak List\|PeakList]] as <tt>hncoO1.peaks</tt>.		#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY Atom List\|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY Peak List\|PeakList]] as <tt>hncoO1.peaks</tt>.
	#Go to next step for [[XEASY Backbone Assignment\|backbone assignment]]		#Go to next step for [[XEASY Backbone Assignment\|backbone assignment]].

			<br>

			An example of the <tt>makeHncoPeak</tt> [[UBNMR\|UBNMR]] script
	An example of the <tt>makeHncoPeak</tt> [[UBNMR\|UBNMR]] script
	<pre>init		<pre>init
	read seq nhsqcO1.seq		read seq nhsqcO1.seq
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	simulate 3D N C -1 2		simulate 3D N C -1 2
	write peaks hncoI1.peaks		write peaks hncoI1.peaks
	</pre>		</pre>
	<br>		<br>

	<br>'''Figure 2: Analysis of the 3D HNNCO by XEASY''' <br>'''A: Before <tt>mr</tt>, showing orthogonal views of each strip'''<br>[[Image:XEASY hnco1.jpg]]		<br>'''Figure 2: Analysis of the 3D HNNCO by XEASY''' <br>'''A: Before <tt>mr</tt>, showing orthogonal views of each strip'''<br>[[Image:XEASY hnco1.jpg]]

	<br>'''B: After <tt>mr</tt>'''<br>[[Image:XEASY hnco2.jpg]] <br>		<br>'''B: After <tt>mr</tt>'''<br>[[Image:XEASY hnco2.jpg]] <br>
			====

	==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====		==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

	#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory<br>		#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory.<br>
	#Run <tt>make32DHncoPeak</tt> in [[UBNMR\|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.		#Run <tt>make32DHncoPeak</tt> in [[UBNMR\|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.
	#Follow the protocol shown above for assignment of 3D HNNCO spectra.<br>		#Follow the protocol shown above for assignment of 3D HNNCO spectra.<br>

			<br>

			An example of the <tt>make32DHncoPeak</tt> [[UBNMR\|UBNMR]] script
	An example of the <tt>make32DHncoPeak</tt> in [[UBNMR\|UBNMR]] script
	<pre>init		<pre>init
	read seq nhsqcO1.seq		read seq nhsqcO1.seq
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	simulate 2D N-pC HN		simulate 2D N-pC HN
	write peaks hncoI1.peaks		write peaks hncoI1.peaks
	</pre>		</pre>
			<br> -- Main.GaohuaLiu - 16 Feb 2007<br>

	-- Main.GaohuaLiu - 16 Feb 2007<br>

Jlmills at 18:31, 24 November 2009

2009-11-24T18:31:06Z

← Older revision		Revision as of 18:31, 24 November 2009
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	#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).		#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
	#Run the <tt>makeHncoPeak</tt> script in [[UBNMR\|UBNMR]].This [[~~XEASY_Peak_List~~\|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[~~XEASY_Peak_List~~\|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.		#Run the <tt>makeHncoPeak</tt> script (see below) in [[UBNMR\|UBNMR]].This [[XEASY Peak List\|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY Peak List\|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
	#Start [[XEASY\|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[~~XEASY_Sequence_List~~\|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[~~XEASY_Atom_List~~\|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[~~XEASY_Peak_List~~\|PeakList]] (<tt>hncoI1.peak</tt>).<br>		#Start [[XEASY\|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY Sequence List\|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY Atom List\|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY Peak List\|PeakList]] (<tt>hncoI1.peak</tt>).<br>
	#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).		#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
	#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).		#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
	#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (<span style="font-family: monospace;">a</span><tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).		#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (<span style="font-family: monospace;">a</span><tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
	#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[~~XEASY_Atom_List~~\|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[~~XEASY_Peak_List~~\|PeakList]] as <tt>hncoO1.peaks</tt>.		#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY Atom List\|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY Peak List\|PeakList]] as <tt>hncoO1.peaks</tt>.
	#Go to next step for [[~~XEASY_Backbone_Assignment~~\|backbone assignment]]		#Go to next step for [[XEASY Backbone Assignment\|backbone assignment]]



			An example of the <tt>makeHncoPeak</tt> [[UBNMR\|UBNMR]] script
			<pre>init
			read seq nhsqcO1.seq
			read prot nhsqcO1.prot
			write prot hncoI1.prot
			simulate 3D N C -1 2
			write peaks hncoI1.peaks
			</pre>
			<br>

	<br>'''Figure 2: Analysis of the 3D HNNCO by XEASY''' <br>'''A: Before <tt>mr</tt>, showing orthogonal views of each strip'''<br>[[Image:XEASY hnco1.jpg]]		<br>'''Figure 2: Analysis of the 3D HNNCO by XEASY''' <br>'''A: Before <tt>mr</tt>, showing orthogonal views of each strip'''<br>[[Image:XEASY hnco1.jpg]]

Jlmills at 18:29, 24 November 2009

2009-11-24T18:29:21Z

@@ Line 46: / Line 46: @@
 ==== '''Analysis of the (3,2)D GFT HNNCO spectrum'''  ====
-*Go to <tt>/protName/analysis/xeasy/hnco</tt>
+#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory<br>
-*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
+#Run <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.
-<blockquote><pre>init
+#Follow the protocol shown above for assignment of 3D HNNCO spectra.<br>
-<br> -- Main.GaohuaLiu - 16 Feb 2007&lt;/pre&gt; <br>

Jlmills at 18:23, 24 November 2009

2009-11-24T18:23:54Z

← Older revision		Revision as of 18:23, 24 November 2009
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	==== '''Analysis of the 3D HNNCO Spectrum''' ====		==== '''Analysis of the 3D HNNCO Spectrum''' ====

	~~#Run the <tt>makeHncoPeak</tt> script in [[UBNMR\|UBNMR]]. A starting peak list for analysis of (3,2)D HNNCO or 3D HNNCO is generated as <tt>hncoI1.peaks</tt>.~~		#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
	~~#In XEASY, perform HNNCO Analysis (described in HNNCO analysis).~~		#Run the <tt>makeHncoPeak</tt> script in [[UBNMR\|UBNMR]].This [[XEASY_Peak_List\|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY_Peak_List\|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		#Start [[XEASY\|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY_Sequence_List\|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY_Atom_List\|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY_Peak_List\|PeakList]] (<tt>hncoI1.peak</tt>).<br>
	~~#In the spectra folder,~~ make a ~~identical~~ copy of HNCO spectrum~~, eg. cp spectrum~~ <tt>HNCO1</tt> ~~to spectrum~~ <tt>HNCO1a</tt>.		#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
	~~#In UBNMR, run~~ <tt>~~makeHncoPeak~~</tt> ~~to produce~~ the ~~file~~ <tt>~~hncoI1.peaks~~</tt>. This ~~<nop>~~PeakList will contain one peak for each amide N,H moiety assigned to SRD-I derived from the 2D [15N,1H]-HSQC ~~<nop>~~PeakList. ~~175 ppm~~ is assigned to ~~all 13C~~' ~~shifts of~~ SRD-II.		#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
	#In XEASY, use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with ~~permutation~~ <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. ~~from <tt>/protName/xeasy/data</tt>; use~~ <tt>ls</tt> to load the ~~sequence file,~~ <tt>nhsqc.seq</tt>~~; use~~ <tt>lc</tt> to load the ~~atom list~~ (~~protlist),~~ <tt>nhsqcO1.prot</tt>~~; use~~ <tt>lp</tt> to load the ~~initial HNCO peaklist from~~ <tt>hncoI1.peak</tt>~~; use~~ <tt>cp</tt> to display the spectrum as a contour plot~~; use~~ <tt>se</tt> ~~to create strips for all of the peaks~~ in the ~~peaklist; use~~ <tt>gs</tt> to ~~display the first set of strips (Figure 2A).~~ <br>~~'''Figure 2: Analysis of the 3D HNNCO by XEASY'''~~ <br>~~'''A: Before~~ <tt>mr</tt>, ~~showing orthogonal views of each strip;'''~~ <br>~~[[Image:XEASY hnco1~~.~~jpg]]~~ <br>		#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (<span style="font-family: monospace;">a</span><tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
	#~~In XEASY, use~~ <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B)~~; use~~ <tt>dp</tt> to delete ~~unobserved~~ predicted peaks (side-chain peaks), and ~~use~~ <tt>pp</tt> manually pick and assign ~~observed unpredicted peaks~~ (~~overlapped in Nhsqc).~~<br>~~'''B: After~~ <tt>mr</tt>~~;''' <br>~~[~~[Image:XEASY hnco2~~.~~jpg]] <br>~~		#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY_Atom_List\|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY_Peak_List\|PeakList]] as <tt>hncoO1.peaks</tt>.
	#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the ~~atom list~~ as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the ~~peaklist~~ as <tt>hncoO1.peaks</tt>.		#Go to next step for [[XEASY_Backbone_Assignment\|backbone assignment]]
	#Go to next step for [[~~XEASY Backbone Assignment~~\|backbone assignment]]
			<br>'''Figure 2: Analysis of the 3D HNNCO by XEASY''' <br>'''A: Before <tt>mr</tt>, showing orthogonal views of each strip'''<br>[[Image:XEASY hnco1.jpg]]

			<br>'''B: After <tt>mr</tt>'''<br>[[Image:XEASY hnco2.jpg]] <br>

	==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====		==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

Jlmills at 18:01, 24 November 2009

2009-11-24T18:01:20Z

← Older revision		Revision as of 18:01, 24 November 2009
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	==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====		==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

	#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[~~fasta~~\|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR\|UBNMR]] to generate an [[XEASY\|XEASY]] [[~~XEASY_Sequence_List~~\|SequenceList]] as <tt>nhsqc.seq</tt> . This [[~~XEASY_Sequence_List~~\|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See XEASY Files for ~~Book~~-keeping: ~~<nop>~~AtomList, ~~<nop>~~SequenceList, ~~<nop>~~PeakList.		#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta\|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR\|UBNMR]] to generate an [[XEASY\|XEASY]] [[XEASY Sequence List\|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List\|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY\|XEASY]] Files for book-keeping: [[XEASY_Atom_List\|AtomList]], [[XEASY_Sequence_List\|SequenceList]], and [[XEASY_Peak_List\|PeakList]].
	#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, XEASY library and ~~<nop>~~SequenceList. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial ~~<nop>~~AtomList is generated automatically~~; use~~ <tt>in</tt> for in-phase 'peak picking' of the 2D [15N,1H]-HSQC spectrum ~~automatically; complete~~ peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to ~~'peak~~ pick' additional peaks (Figure.1A); Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C)~~; use~~ <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save the ~~updated <nop>~~AtomList as <tt>nhsqcO1.prot</tt> and ~~<nop>PeakList as~~ <tt>~~nhsqcO1.peaks~~</tt>. Then, 15N / ~~amide~~ 1HN chemical shifts are transferred into the ~~<nop>~~AtomList entries corresponding to '''SRD-I'''. At times the <tt>ar</tt> command gives an error "Atom N 201 not known!". This can be rectified by loading a different library file [ll] from /nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib and then trying to auto assign the peaks. <br><br>'''Figure 1.''' Peak picking the NHSQC spectrum*		#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY\|XEASY]] library and [[XEASY_Sequence_List\|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY_Atom_List\|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY_Peak_List\|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY_Atom_List\|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY_Atom_List\|AtomList]] entries corresponding to '''SRD-I'''.

	'''~~A: After in-phase peak picking;~~ ''' ~~<br>~~ [~~[Image:XEASY hsqc2.jpg~~]~~] <br>~~		<br><br>'''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

	'''B: XEASY <tt>ar<~~/tt~~> ~~window'''~~		'''A: After in-phase peak picking'''<br> [[Image:XEASY hsqc2.jpg]] <br>
			<br>

	<br> [[Image:XEASY hsqc ar.jpg]]<br>		'''B: XEASY <tt>ar</tt> window'''<br> [[Image:XEASY hsqc ar.jpg]]<br>

	<br> '''C: After ~~XEASY command~~ <tt>ar</tt> .'''		<br> '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>'''<br>

	<br>		[[Image:XEASY hsqc3.jpg]]<br>

	~~[[Image:XEASY hsqc3.jpg]]<br>~~

	~~#In UBNMR run <tt>makeHncoPeak</tt>. A starting peak list for analysis of (3,2)D HNNCO or 3D HNNCO is generated as <tt>hncoI1.peaks</tt>. /~~
	~~#In XEASY, perform HNNCO Analysis (described in HNNCO analysis).~~

	==== '''Analysis of the 3D HNNCO Spectrum''' ====		==== '''Analysis of the 3D HNNCO Spectrum''' ====

			#Run the <tt>makeHncoPeak</tt> script in [[UBNMR\|UBNMR]]. A starting peak list for analysis of (3,2)D HNNCO or 3D HNNCO is generated as <tt>hncoI1.peaks</tt>.
			#In XEASY, perform HNNCO Analysis (described in HNNCO analysis).
	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		#Go to <tt>/protName/analysis/xeasy/hnco</tt>
	#In the spectra folder, make a identical copy of HNCO spectrum, eg. cp spectrum <tt>HNCO1</tt> to spectrum <tt>HNCO1a</tt>.		#In the spectra folder, make a identical copy of HNCO spectrum, eg. cp spectrum <tt>HNCO1</tt> to spectrum <tt>HNCO1a</tt>.

Jlmills at 16:53, 24 November 2009

2009-11-24T16:53:24Z

← Older revision		Revision as of 16:53, 24 November 2009
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	== Spin System Identification ==		== Spin System Identification ==

	2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In ~~Xeasy~~-based ~~GFT~~ approach spin systems are called '''SRD''' spin systems.		2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY\|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

	(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by '''CSI''' and '''TALOS''' ~~programs~~.		(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI\|'''CSI''']] and [[TALOS\|'''TALOS''']].

	=== '''Spin System Identification with XEASY/UBNMR''' ===		=== '''Spin System Identification with XEASY/UBNMR''' ===
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	==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====		==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

	#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino ~~acids~~ sequence in FASTA format, obtained, for example, from the SPINE web page, and save it as <tt>aa.seq</tt>. ~~In UBNMR, run~~ <tt>makeSeq</tt>~~. This generates~~ an XEASY ~~<nop>~~SequenceList as <tt>nhsqc.seq</tt> . This ~~<nop>~~SequenceList contains entries for the amino acids residues first, followed by two sets of ~~SRDs~~ named in the following '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See XEASY Files for Book-keeping: <nop>AtomList, <nop>SequenceList, <nop>PeakList.		#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[fasta\|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR\|UBNMR]] to generate an [[XEASY\|XEASY]] [[XEASY_Sequence_List\|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY_Sequence_List\|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See XEASY Files for Book-keeping: <nop>AtomList, <nop>SequenceList, <nop>PeakList.
	#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, XEASY library and <nop>SequenceList. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial <nop>AtomList is generated automatically; use <tt>in</tt> for in-phase 'peak picking' of the 2D [15N,1H]-HSQC spectrum automatically; complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to 'peak pick' additional peaks (Figure.1A); Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C); use <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save the updated <nop>AtomList as <tt>nhsqcO1.prot</tt> and <nop>PeakList as <tt>nhsqcO1.peaks</tt>. Then, 15N / amide 1HN chemical shifts are transferred into the <nop>AtomList entries corresponding to '''SRD-I'''. At times the <tt>ar</tt> command gives an error "Atom N 201 not known!". This can be rectified by loading a different library file [ll] from /nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib and then trying to auto assign the peaks. <br><br>'''Figure 1.''' Peak picking the NHSQC spectrum*		#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, XEASY library and <nop>SequenceList. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial <nop>AtomList is generated automatically; use <tt>in</tt> for in-phase 'peak picking' of the 2D [15N,1H]-HSQC spectrum automatically; complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to 'peak pick' additional peaks (Figure.1A); Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C); use <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save the updated <nop>AtomList as <tt>nhsqcO1.prot</tt> and <nop>PeakList as <tt>nhsqcO1.peaks</tt>. Then, 15N / amide 1HN chemical shifts are transferred into the <nop>AtomList entries corresponding to '''SRD-I'''. At times the <tt>ar</tt> command gives an error "Atom N 201 not known!". This can be rectified by loading a different library file [ll] from /nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib and then trying to auto assign the peaks. <br><br>'''Figure 1.''' Peak picking the NHSQC spectrum*

	'''A: After in-phase peak picking; '''		'''A: After in-phase peak picking; ''' <br> [[Image:XEASY hsqc2.jpg]] <br>
	<br>
	[[Image:XEASY hsqc2.jpg]] <br>

	'''B: XEASY <tt>ar</tt> window'''		'''B: XEASY <tt>ar</tt> window'''

	<br>		<br> [[Image:XEASY hsqc ar.jpg]]<br>
	[[Image:XEASY hsqc ar.jpg]]<br>

			<br> '''C: After XEASY command <tt>ar</tt> .'''
	'''C: After XEASY command <tt>ar</tt> .'''

	<br>		<br>
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	*Go to <tt>/protName/analysis/xeasy/hnco</tt>		*Go to <tt>/protName/analysis/xeasy/hnco</tt>
	*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>		*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
	<blockquote><pre>		<blockquote><pre>init
	init
	read seq nhsqc.seq		read seq nhsqc.seq
	read prot noe.prot		read prot noe.prot

Agutmana at 19:06, 10 November 2009

2009-11-10T19:06:02Z

← Older revision		Revision as of 19:06, 10 November 2009
Line 47:		Line 47:
	==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====		==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		*Go to <tt>/protName/analysis/xeasy/hnco</tt>
	#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>		*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
	<blockquote><~~code~~>init		<blockquote><pre>
	read seq nhsqc.seq		init
	read prot noe.prot		read seq nhsqc.seq
	write prot hnco.prot		read prot noe.prot

			write prot hnco.prot
	simulate 2D N+pC HN		simulate 2D N+pC HN
	simulate 2D N-pC HN		simulate 2D N-pC HN
	write peaks HNNCO.peaks</~~code~~></blockquote>		write peaks HNNCO.peaks
	#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.		</pre></blockquote>
			*Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

	<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>		<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>

Agutmana at 19:05, 10 November 2009

2009-11-10T19:05:05Z

← Older revision		Revision as of 19:05, 10 November 2009
Line 49:		Line 49:
	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		#Go to <tt>/protName/analysis/xeasy/hnco</tt>
	#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>		#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
	<blockquote><~~nowiki~~>init		<blockquote><code>init
	read seq nhsqc.seq		read seq nhsqc.seq
	read prot noe.prot		read prot noe.prot
Line 55:		Line 55:
	simulate 2D N+pC HN		simulate 2D N+pC HN
	simulate 2D N-pC HN		simulate 2D N-pC HN
	write peaks HNNCO.peaks</~~nowiki~~></blockquote>		write peaks HNNCO.peaks</code></blockquote>
	#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.		#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

	<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>		<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>

Agutmana at 19:04, 10 November 2009

2009-11-10T19:04:12Z

← Older revision		Revision as of 19:04, 10 November 2009
Line 49:		Line 49:
	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		#Go to <tt>/protName/analysis/xeasy/hnco</tt>
	#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>		#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
	<blockquote><~~pre~~>init		<blockquote><nowiki>init
	read seq nhsqc.seq		read seq nhsqc.seq
	read prot noe.prot		read prot noe.prot
Line 55:		Line 55:
	simulate 2D N+pC HN		simulate 2D N+pC HN
	simulate 2D N-pC HN		simulate 2D N-pC HN
	write peaks HNNCO.peaks</~~pre~~></blockquote>		write peaks HNNCO.peaks</nowiki></blockquote>
	#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.		#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

	<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>		<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>

Agutmana at 19:01, 10 November 2009

2009-11-10T19:01:22Z

← Older revision		Revision as of 19:01, 10 November 2009
Line 49:		Line 49:
	#Go to <tt>/protName/analysis/xeasy/hnco</tt>		#Go to <tt>/protName/analysis/xeasy/hnco</tt>
	#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>		#In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist.<br>
	<pre>		<blockquote><pre>init
	init
	read seq nhsqc.seq		read seq nhsqc.seq
	read prot noe.prot		read prot noe.prot

	write prot hnco.prot		write prot hnco.prot
	simulate 2D N+pC HN		simulate 2D N+pC HN
	simulate 2D N-pC HN		simulate 2D N-pC HN
	write peaks HNNCO.peaks</pre><br>~~<br>~~		write peaks HNNCO.peaks</pre></blockquote>

	#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.		#Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

			<br> -- Main.GaohuaLiu - 16 Feb 2007</pre> <br>
	-- Main.GaohuaLiu - 16 Feb 2007</pre>
	<br>