Resonance Assignment/CARA/Backbone assignment - Revision history

Alex at 02:17, 9 December 2009

2009-12-09T02:17:25Z

@@ Line 33: / Line 33: @@
 *Before linking spin systems, make sure that peak shapes in 1D slices match for all such spectra. You may need to zoom in on individual peaks, since 1D slices are automatically scaled to the maximum amplitude in the zoomed region.
 *The '''Show Alignment...''' window may display no match if you have highly unusual chemical shifts in your spin system. This is more likely if you include proton spins, such as HA-1 and HB-1 from 3D HBHA(CO)NH.
 *Special attention is needed when assigning and unassigning spin systems. Unassigning a spin system that is part of a linked fragment will remove assignments of all spin systems in that fragment. Conversely, assigning a spin system to a particular residue will automatically assign other spin systems in the same fragment; if the newly assigned fragment overlaps with some previously assigned fragments, they will be unassigned.
 *It is thus safer to use '''Show Free Successors''' and '''Show Free Predecessors''' for routine assignments. Invoke '''Show All Successors''' and '''Show All Predecessors''' only if you cannot find matches or have reasons to suspect that existing assignments may be incorrect.
+*To unlink spin systems right-click on a spin system tab and select '''Unlink Successor''' or '''Unlink Predecessor''' from the context menu.
 == Semi-automated resonance assignment ==
-Semi-automated resonance assignment is usually faster than purely manual approach.
+Semi-automated resonance assignment is usually faster than purely manual approach. Automated methods almost never yield absolutely complete and accurate results and thus require manual inspection an finalizing by an expert.
-=== Using PINE server ===
+=== Backbone Assignment with PINE ===
 First, you would need to prepare simulated peaklists in XEASY format for a set of 3D triple-resonance experiments (for example, CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO and HBHA(CO)NH ). You can use the '''Pick_3D_Peaks''' LUA script, however, can only pick spins and does not generate peaklists directly.
@@ Line 60: / Line 62: @@
 Please note, that spin systems not assigned by PINE will not be imported back and will have to picked again. This is not a big issue as long as the automated assignments are fairly complete.
-=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra'''  ===
+=== Backbone Assignment with AutoAssign  ===
 Once CA, CA-1, CB, and CB-1 spins have been created you can run AutoAssign.
@@ Line 111: / Line 83: @@
 The newer version of AutoAssign should be able to handle GFT shifts directly, and this protocol may need to be modified to take advantage of it.
-[http://www.autolink.nmr-software.org/ AutoLink] is an alternative automated backbone assignment package written in LUA specifically for CARA by Jim Masse.
+[http://www.autolink.nmr-software.org/AutoLink] is an alternative automated backbone assignment package written in LUA specifically for CARA by Jim Masse.
 <br> -- Main.AlexEletski - 06 Jul 2007

Alex: /* Using PINE server */

2009-12-09T02:02:18Z

Using PINE server

← Older revision		Revision as of 02:02, 9 December 2009
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	#Verify assignments made by PINE and complete the assignment manually as described above.		#Verify assignments made by PINE and complete the assignment manually as described above.

	Please note that spin systems not assigned by PINE will not be imported back and will have to picked again. This is not a big issue as long as the automated assignments are fairly complete.		Please note, that spin systems not assigned by PINE will not be imported back and will have to picked again. This is not a big issue as long as the automated assignments are fairly complete.

	=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===

Alex: /* Using PINE server */

2009-12-09T02:01:56Z

Using PINE server

← Older revision		Revision as of 02:01, 9 December 2009
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	=== Using PINE server ===		=== Using PINE server ===

	First you would need to prepare simulated peaklists in XEASY format for a set of 3D triple-resonance experiments (for example, CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO and HBHA(CO)NH ). You can use the '''Pick_3D_Peaks''' LUA script, however, can only pick spins and does not generate peaklists directly.		First, you would need to prepare simulated peaklists in XEASY format for a set of 3D triple-resonance experiments (for example, CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO and HBHA(CO)NH ). You can use the '''Pick_3D_Peaks''' LUA script, however, can only pick spins and does not generate peaklists directly.

	#Run '''Pick_3D_Peaks''' LUA script to pick spins in a 3D spectrum, such as CBCA(CO)NH. Use an appropriate generic spin label (for example, ?CA-1 would work well for CBCA(CO)NH).		#Run '''Pick_3D_Peaks''' LUA script to pick spins in a 3D spectrum, such as CBCA(CO)NH. Use an appropriate generic spin label (for example, ?CA-1 would work well for CBCA(CO)NH).
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	#In PolyScope click '''File''' -> '''Export strip peaks to MonoScope...''' - this will open a MonoScope window with a simulated peaklist.		#In PolyScope click '''File''' -> '''Export strip peaks to MonoScope...''' - this will open a MonoScope window with a simulated peaklist.
	#In the MonoScope window, add peaklist to repository and update the amplitudes. Run CopyAmpToVol LUA script to set peak volumes. In the MonoScope window export the peaklist as XEASY format.		#In the MonoScope window, add peaklist to repository and update the amplitudes. Run CopyAmpToVol LUA script to set peak volumes. In the MonoScope window export the peaklist as XEASY format.
	#Delete the newly picked ?CA-1 spins by executing the RemoveSpins LUA script.		#Delete the newly picked ?CA-1 spins by executing the RemoveSpins LUA script (otherwise they will automatically show up in HNCACB, for example).
	#Repeat for other 3D triple-resonance spectra.		#Repeat for other 3D triple-resonance spectra that you want to use.
			#Export 2D [15N,1H] HSQC peaklist useing '''File''' -> '''Export plane peaks to MonoScope...''' in PolyScope.
			#Peaklists exported from CARA in XEASY format will carry spin system numbers in the form of comments on separate lines that cannot be properly read by PINE. Remove these lines with <tt>grep -v "# " hnco.peaks > hnco2.peaks</tt> command in a UNIX shell.
			#Submit the data to PINE server and,
			#If satisfied with the results then delete all spins and spin systems and import the assignments from the <tt>.prot</tt> in PINE output by right-clicking on your project '''Import''' -> '''Atom List''' in the main window of CARA. Don't forget to back up your repository under a different filename!
			#Correct HB assignments in alanines. Due to a bug in PINE they are imported as HB1, HB2 and HB3 instead of HB.
			#Verify assignments made by PINE and complete the assignment manually as described above.

	~~To export 2D [15N,1H] HSQC peak use '''File''' -> '''Export plane peaks to MonoScope...'''~~		Please note that spin systems not assigned by PINE will not be imported back and will have to picked again. This is not a big issue as long as the automated assignments are fairly complete.

	~~Peaklists exported from CARA in XEASY format~~ will ~~carry spin system numbers as comments on separate lines. Remove these line with <tt>grep -v "# " hnco.peaks > hnco2.peaks</tt>.~~

	~~Submit the data~~ to ~~PINE server~~. ~~If the extent of assignments~~ is ~~nearly complete you can import the assignments.~~

	~~#Back up your repository under~~ a ~~different filename. Delete all spins and spin systems.~~
	~~#Import chemical shifts from the <tt>.prot</tt> in PINE output by right-clicking on your project '''Import''' -> '''Atom List'''.~~

	~~Verify~~ the assignments ~~made by PINE and~~ complete ~~the assignment manually as described above~~.

	=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===

Alex: moved Backbone Assignment with CARA to Resonance Assignment/CARA/Backbone assignment

2009-12-09T01:47:00Z

moved Backbone Assignment with CARA to Resonance Assignment/CARA/Backbone assignment

← Older revision	Revision as of 01:47, 9 December 2009
(No difference)

Alex at 16:43, 8 December 2009

2009-12-08T16:43:52Z

@@ Line 14: / Line 14: @@
 Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope
-== Picking spin systems manually ==
+== Manual resonance assignment ==
 #In PolyScope or SynchroScope select a spin system by left-clicking in the 2D [15N,1H] HSQC plane, or use '''View -&gt; Goto System'''. This will update the displayed strips.
@@ Line 24: / Line 26: @@
 Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope
-== Linking and assigning spin systems ==
+=== Linking and assigning ===
 Once you have picked all the spin systems you can assign them to specific residues. This is achieved by building fragments of linked spin-systems and mapping them to the protein sequence. The procedure is outlined quite well in http://wiki.cara.nmr.ch/StripScope. Additional issues to consider:
@@ Line 35: / Line 37: @@
 === '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra'''  ===

Alex: /* Linking and assigning spin systems */

2009-12-08T15:25:59Z

Linking and assigning spin systems

← Older revision		Revision as of 15:25, 8 December 2009
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	Once you have picked all the spin systems you can assign them to specific residues. This is achieved by building fragments of linked spin-systems and mapping them to the protein sequence. The procedure is outlined quite well in http://wiki.cara.nmr.ch/StripScope. Additional issues to consider:		Once you have picked all the spin systems you can assign them to specific residues. This is achieved by building fragments of linked spin-systems and mapping them to the protein sequence. The procedure is outlined quite well in http://wiki.cara.nmr.ch/StripScope. Additional issues to consider:

	*Use StripScope to display appropriate spectra that show both sequential and intra-residue ~~correlation~~, such as HNCA, HNCACB, HN(CA)CO, 15N-resolved NOESY.		*Use StripScope to display appropriate spectra that show both sequential and intra-residue correlations, such as HNCA, HNCACB, HN(CA)CO, 15N-resolved NOESY.
	*		*Before linking spin systems, make sure that peak shapes in 1D slices match for all such spectra. You may need to zoom in on individual peaks, since 1D slices are automatically scaled to the maximum amplitude in the zoomed region.
			*The '''Show Alignment...''' window may display no match if you have highly unusual chemical shifts in your spin system. This is more likely if you include proton spins, such as HA-1 and HB-1 from 3D HBHA(CO)NH.
	~~#Select~~		*Special attention is needed when assigning and unassigning spin systems. Unassigning a spin system that is part of a linked fragment will remove assignments of all spin systems in that fragment. Conversely, assigning a spin system to a particular residue will automatically assign other spin systems in the same fragment; if the newly assigned fragment overlaps with some previously assigned fragments, they will be unassigned.
	~~#Display each~~ linked fragment of spin systems ~~by right-clicking on~~ a spin system and ~~selecting~~ '''Show ~~Fragment~~''' ~~from the context menu~~.		*It is thus safer to use '''Show Free Successors''' and '''Show Free Predecessors''' for routine assignments. Invoke '''Show All Successors''' and '''Show All Predecessors''' only if you cannot find matches or have reasons to suspect that existing assignments may be incorrect.
	~~#Inspect sequential connectives between strips.~~
	~~#Check how well the fragment fits the sequence by right-clicking on a spin system~~ and ~~selecting~~ '''Show ~~Alignment~~''' ~~from the context menu~~.

Alex: /* Picking spin systems manually */

2009-12-08T15:02:10Z

Picking spin systems manually

← Older revision		Revision as of 15:02, 8 December 2009
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	#In PolyScope or SynchroScope select a spin system by left-clicking in the 2D [15N,1H] HSQC plane, or use '''View -> Goto System'''. This will update the displayed strips.		#In PolyScope or SynchroScope select a spin system by left-clicking in the 2D [15N,1H] HSQC plane, or use '''View -> Goto System'''. This will update the displayed strips.
	#In the strip panel set the cursor on a peak and ~~use~~ '''~~right click ->~~ Pick Label...''' or '''~~right click ->~~ Pick Spin''' and '''~~right click ->~~ Label Spin''' to pick C-1. Repeat ~~last two steps~~ for all spin systems.		#In the strip panel set the cursor on a peak, right-click, and select '''Pick Label...''', or '''Pick Spin''' and '''Label Spin...''' to pick the appropriate spin (e.g., C-1 in HNCO).
			#Repeat for all spin systems.

	For example, if you are working with 3D HNCO, HN(CA)CO, CBCA(CO)NH and HNCACB spectra, you would first pick all C-1 spins in HNCO. Then you can switch the spectrum to HN(CA)CO and all C-1 spins will be displayed, allowing you to assign the remaining spins to C spins. You can follow the same procedure by picking CA-1 and CB-1 spins in CBCA(CO)NH first, then picking CA and CB in HNCACB.		For example, if you are working with 3D HNCO, HN(CA)CO, CBCA(CO)NH and HNCACB spectra, you would first pick all C-1 spins in HNCO. Then you can switch the spectrum to HN(CA)CO and all C-1 spins will be displayed, allowing you to assign the remaining spins to C spins. You can follow the same procedure by picking CA-1 and CB-1 spins in CBCA(CO)NH first, then picking CA and CB in HNCACB.

	Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope		Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope

	== Linking and assigning spin systems ==		== Linking and assigning spin systems ==

Alex at 14:58, 8 December 2009

2009-12-08T14:58:13Z

← Older revision		Revision as of 14:58, 8 December 2009
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			Here we speak of backbone assignment in a broad sense, including assignment of CB and HB spins. Generally the approach outlined below can be applied to various set of 3D triple-resonance experiments.

	== Initializing spin systems ==		== Initializing spin systems ==

Alex at 19:29, 6 December 2009

2009-12-06T19:29:13Z

← Older revision		Revision as of 19:29, 6 December 2009
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	== ~~'''~~Initializing spin systems~~'''~~ ==		== Initializing spin systems ==

	Normally you would start picking new spins systems from a 2D [<sup>15</sup>N,<sup>1</sup>H] HSQC. The quickest way is to initialize them automatically.		Normally you would start picking new spins systems from a 2D [<sup>15</sup>N,<sup>1</sup>H] HSQC. The quickest way is to initialize them automatically.
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	After initializing spin systems you can proceed to pick 3D spectra either manually or automatically.		After initializing spin systems you can proceed to pick 3D spectra either manually or automatically.

	== ~~'''~~Picking spin systems manually~~'''~~ ==		Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope

			== Picking spin systems manually ==

	#In PolyScope or SynchroScope select a spin system by left-clicking in the 2D [15N,1H] HSQC plane, or use '''View -> Goto System'''. This will update the displayed strips.		#In PolyScope or SynchroScope select a spin system by left-clicking in the 2D [15N,1H] HSQC plane, or use '''View -> Goto System'''. This will update the displayed strips.
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	Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope		Also see instructions on how to use SynchroScope here: http://www.cara.ethz.ch/Wiki/SynchroScope

	~~<br>~~		== Linking and assigning spin systems ==

			Once you have picked all the spin systems you can assign them to specific residues. This is achieved by building fragments of linked spin-systems and mapping them to the protein sequence. The procedure is outlined quite well in http://wiki.cara.nmr.ch/StripScope. Additional issues to consider:

			*Use StripScope to display appropriate spectra that show both sequential and intra-residue correlation, such as HNCA, HNCACB, HN(CA)CO, 15N-resolved NOESY.
			*

			#Select
			#Display each linked fragment of spin systems by right-clicking on a spin system and selecting '''Show Fragment''' from the context menu.
			#Inspect sequential connectives between strips.
			#Check how well the fragment fits the sequence by right-clicking on a spin system and selecting '''Show Alignment''' from the context menu.


	~~<br>~~

	=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===		=== '''Picking Spins in (4,3)D GFT HNNCABCA and CABCA(CO)NHN Spectra''' ===

Alex at 18:53, 6 December 2009

2009-12-06T18:53:53Z

Show changes