Resonance Assignment/CARA/Aliphatic side-chain assignment - Revision history

Xianzhon: /* Filling in the gaps */

2012-08-31T14:21:27Z

Filling in the gaps

← Older revision		Revision as of 14:21, 31 August 2012
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	== Filling in the gaps ==		== Filling in the gaps ==

	After the first pass through the sequence some residues ~~that~~ were skipped. You may also have small gaps in the backbone assignment.		After the first pass through the sequence some residues were skipped. You may also have small gaps in the backbone assignment.

	One category of skipped residues are those directly preceding prolines, since they will have CA and CB assignments at most. According to the alternative approach you have to find HA and HB in the H strip is the same residue in 15N-resolved NOESY (windows 6 and 7) and in the HD2 and HD3 strips of the succeeding Pro in 13C-resolved NOESY (window 3). Note that the latter is only valid for ''trans'' peptide bond. You should consider looking at the HA strip of Pro, if you don't any candidates in HD2 and HD3 strips.		One category of skipped residues are those directly preceding prolines, since they will have CA and CB assignments at most. According to the alternative approach you have to find HA and HB in the H strip is the same residue in 15N-resolved NOESY (windows 6 and 7) and in the HD2 and HD3 strips of the succeeding Pro in 13C-resolved NOESY (window 3). Note that the latter is only valid for ''trans'' peptide bond. You should consider looking at the HA strip of Pro, if you don't see any candidates in HD2 and HD3 strips.

	-- Main.AlexEletski - 09 Mar 2007		-- Main.AlexEletski - 09 Mar 2007

Alex at 19:48, 9 December 2009

2009-12-09T19:48:22Z

← Older revision		Revision as of 19:48, 9 December 2009
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			== Introduction ==

	For a short overview of heteronuclear side-chain assignment see this page:		For a short overview of heteronuclear side-chain assignment see this page:
	http://www.cara.ethz.ch/Wiki/HeteronuclearSidechainAssignment		http://www.cara.ethz.ch/Wiki/HeteronuclearSidechainAssignment

Alex at 19:47, 9 December 2009

2009-12-09T19:47:43Z

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Alex: moved Aliphatic Side Chain Assignment with CARA to Resonance Assignment/CARA/Aliphatic side-chain assignment

2009-12-09T18:53:54Z

moved Aliphatic Side Chain Assignment with CARA to Resonance Assignment/CARA/Aliphatic side-chain assignment

← Older revision	Revision as of 18:53, 9 December 2009
(No difference)

Agutmana at 22:33, 9 November 2009

2009-11-09T22:33:35Z

← Older revision		Revision as of 22:33, 9 November 2009
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	Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.		Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.

	=== '''Working with SystemScope windows''' ===		=== '''Working with SystemScope windows''' ===

	To display a strip of interest in a StripScope window, select a residue from the pull-down menu. Spins belonging to the selected spins systems will appear in the panel below it. In the second panel below, a list of "strip anchors" (e.g. CA/HA and CB/HB pairs) will be displayed. Double-click on a spin pair, or right-click and select '''Show Strip'''. A corresponding strip will be displayed to the right. Its dimensions are '''X* and *Y''', as defined by the rotation of the spectrum.		To display a strip of interest in a StripScope window, select a residue from the pull-down menu. Spins belonging to the selected spins systems will appear in the panel below it. In the second panel below, a list of "strip anchors" (e.g. CA/HA and CB/HB pairs) will be displayed. Double-click on a spin pair, or right-click and select '''Show Strip'''. A corresponding strip will be displayed to the right. Its dimensions are '''X '''and'''Y''', as defined by the rotation of the spectrum.

	If you are opening a 3D 13C-resolved NOESY, 3D (H)CCH-COSY or 3D (H)CCH-TOCSY for the first time, you would want to change the "strip widths" (default is 1 ppm). Right-click on a displayed strip and select '''Set Peak Width'''. Typical values should be 2-3 ppm (13C width in 3D (H)CCH experiments), and 0.3-0.5 ppm (1H width in 3D 13C-resolved NOESY).		If you are opening a 3D 13C-resolved NOESY, 3D (H)CCH-COSY or 3D (H)CCH-TOCSY for the first time, you would want to change the "strip widths" (default is 1 ppm). Right-click on a displayed strip and select '''Set Peak Width'''. Typical values should be 2-3 ppm (13C width in 3D (H)CCH experiments), and 0.3-0.5 ppm (1H width in 3D 13C-resolved NOESY).

	If you right-click in the strip panel and select '''Show Depth''', a spectral plane orthogonal to the strip (with dimensions '''Z* and *Y''') will be displayed to the right. You may want to zoom the plane out by right-clicking on it an selecting '''Fit window''', or left-clicking in it and using the <tt>ww</tt> keyboard shortcut.		If you right-click in the strip panel and select '''Show Depth''', a spectral plane orthogonal to the strip (with dimensions '''Z '''and'''Y''') will be displayed to the right. You may want to zoom the plane out by right-clicking on it an selecting '''Fit window''', or left-clicking in it and using the <tt>ww</tt> keyboard shortcut.

	You can also select a spin in the strip panel and display the orthogonal plane taken at that spin's position by right-clicking and selecting '''Show Orthogonal'''. It is possible to select several spins at once, and thus display more than one plane.		You can also select a spin in the strip panel and display the orthogonal plane taken at that spin's position by right-clicking and selecting '''Show Orthogonal'''. It is possible to select several spins at once, and thus display more than one plane.

	SystemScope in window 5 is used to verify whether diasterotopic spin pairs are degenerate, and pick a missing spin, if not. For example, you have HB/CB of Asn assigned. Display this HB/HB strip in window 5 and use right-click -> '''Show Depth'''. In the depth plane zoom into the region around HB. If HB are non-degenerate and you have missed the other spin, you would see two strong HB2-HB3 peaks both symmetric with respect to diagonal. In addition, the two spin positions should exhibit very similar (though not necessarily identical) NOE peak patterns along Y, including HB-HA and HB-H peaks.		SystemScope in window 5 is used to verify whether diasterotopic spin pairs are degenerate, and pick a missing spin, if not. For example, you have HB/CB of Asn assigned. Display this HB/HB strip in window 5 and use right-click -> '''Show Depth'''. In the depth plane zoom into the region around HB. If HB are non-degenerate and you have missed the other spin, you would see two strong HB2-HB3 peaks both symmetric with respect to diagonal. In addition, the two spin positions should exhibit very similar (though not necessarily identical) NOE peak patterns along Y, including HB-HA and HB-H peaks.

	=== '''Calibration''' ===		=== '''Calibration''' ===

Agutmana at 22:30, 9 November 2009

2009-11-09T22:30:00Z

Agutmana: moved NESG:CARAAliphaticSideChainAssignment to Aliphatic Side Chain Assignment with CARA

2009-11-06T17:12:26Z

moved NESG:CARAAliphaticSideChainAssignment to Aliphatic Side Chain Assignment with CARA

← Older revision	Revision as of 17:12, 6 November 2009
(No difference)

Admin: Created page with ' == '''Aliphatic Side-chain Resonance Assignment''' == === '''Introduction''' === For a short overview of heteronuclear side-chain assignment see this page: http://www.cara.…'

2009-10-09T17:48:19Z

Created page with ' == '''Aliphatic Side-chain Resonance Assignment''' == === '''Introduction''' === For a short overview of heteronuclear side-chain assignment see this page: http://www.cara.…'

Show changes

← Older revision		Revision as of 22:30, 9 November 2009
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	Also, note that the statistics can be misleading for diastereotopic protons and methyl groups of Val and Leu. Even though HB2 and HB3 of Phe have very close average values, in a real spectrum they can be far apart. The same is true for CG1/2 and HG1/2 of Val, as well as CD1/2 and HD1/2 of Leu.		Also, note that the statistics can be misleading for diastereotopic protons and methyl groups of Val and Leu. Even though HB2 and HB3 of Phe have very close average values, in a real spectrum they can be far apart. The same is true for CG1/2 and HG1/2 of Val, as well as CD1/2 and HD1/2 of Leu.

	=== '''Displaying spectra in multiple windows''' ===		=== '''Displaying spectra in multiple windows''' ===

	The complete set for aliphatic side chain assignment has 7 windows open at the same time:		The complete set for aliphatic side chain assignment has 7 windows open at the same time:

	# Open 3D (H)CCH-COSY with '''Open SystemScope (rotated)...'''. In the rotation matrix pop-up window choose the following orientation: '''Hinept''' dimension must be '''Z, Ccosy''' must be '''Y, and Cinept''' must be X. Thus, the left panel of SystemScope will contain a C-C plane (with Ccosy dimension vertical), and the right panel will contain the H-C plane. This issue is addressed in detail in this FAQ question: http://www.cara.ethz.ch/Wiki/FAQ#III10		#Open 3D (H)CCH-COSY with '''Open SystemScope (rotated)...'''. In the rotation matrix pop-up window choose the following orientation: '''Hinept''' dimension must be '''Z, Ccosy''' must be '''Y, and Cinept''' must be X. Thus, the left panel of SystemScope will contain a C-C plane (with Ccosy dimension vertical), and the right panel will contain the H-C plane. This issue is addressed in detail in this FAQ question: http://www.cara.ethz.ch/Wiki/FAQ#III10
	# Open 3D (H)CCH-TOCSY with '''Open SystemScope (rotated)...''' in the same manner as (H)CCH-COSY. Though side-chain assignment can be performed with (H)CCH-COSY, the use of 3D (H)CCH-TOCSY is recommended to improve reliability, especially in case of low signal-to-noise and severe overlap.		#Open 3D (H)CCH-TOCSY with '''Open SystemScope (rotated)...''' in the same manner as (H)CCH-COSY. Though side-chain assignment can be performed with (H)CCH-COSY, the use of 3D (H)CCH-TOCSY is recommended to improve reliability, especially in case of low signal-to-noise and severe overlap.
	# Open aliphatic 2D [13C, 1H]-HSQC with '''Open PolyScope''' and select aliphatic 13C-resolved NOESY for the strip display. This is similar to displaying 3D HNCO or 3D 15N-resloved NOESY paired with a 2D [15N, 1H]-HSQC.		#Open aliphatic 2D [13C, 1H]-HSQC with '''Open PolyScope''' and select aliphatic 13C-resolved NOESY for the strip display. This is similar to displaying 3D HNCO or 3D 15N-resloved NOESY paired with a 2D [15N, 1H]-HSQC.
	# Open 3D 13C-resolved NOESY with '''Open SystemScope'''. Rotation is normally not needed, since the default dimension order is '''Hinept''' = '''X, Hnoe''' = '''Y, Cinept''' = Z. Thus, the left panel of SystemScope will contain an H-H plane (with Hnoe dimension vertical), and the right panel will contain the H-C plane.		#Open 3D 13C-resolved NOESY with '''Open SystemScope'''. Rotation is normally not needed, since the default dimension order is '''Hinept''' = '''X, Hnoe''' = '''Y, Cinept''' = Z. Thus, the left panel of SystemScope will contain an H-H plane (with Hnoe dimension vertical), and the right panel will contain the H-C plane.
	# Open 3D 13C-resolved NOESY with '''Open SystemScope (rotated)...'''. In the rotation matrix pop-up window choose the following orientation: '''Cinept''' = '''X, Hnoe''' = '''Y, and Hinept''' must be Z.		#Open 3D 13C-resolved NOESY with '''Open SystemScope (rotated)...'''. In the rotation matrix pop-up window choose the following orientation: '''Cinept''' = '''X, Hnoe''' = '''Y, and Hinept''' must be Z.
	# Open 3D 15N-resolved NOESY with '''Open StripScope'''. This window will also be used to display triple-resonance spectra.		#Open 3D 15N-resolved NOESY with '''Open StripScope'''. This window will also be used to display triple-resonance spectra.
	# Open 2D [15N, 1H]-HSQC with '''Open PolyScope''' and select 15N-resolved NOESY for the strip display. This window will also be used to display triple-resonance spectra (like HBHA(CO)NH).		#Open 2D [15N, 1H]-HSQC with '''Open PolyScope''' and select 15N-resolved NOESY for the strip display. This window will also be used to display triple-resonance spectra (like HBHA(CO)NH).

	It is recommended to disable the default automatic contour level adjustment . In SystemScope windows uncheck '''View->Auto Contour Level''' and set the appropriate base contour level, '''View->Set Contour Parameters'''. In PolyScope uncheck '''Plane->Auto Contour Level''' and '''Strip->Auto Contour Level''', and use '''Plane->Set Contour Parameters''' and '''Strip->Set Contour Parameters'''. The last base contour level setting will be recalled the next time CARA is started. Spectra transformed with PROSA will usually be scaled so that RMS noise is 100, therefore the common base level for such spectra is 400. Spectra transformed with other software may need a different base level.		It is recommended to disable the default automatic contour level adjustment . In SystemScope windows uncheck '''View->Auto Contour Level''' and set the appropriate base contour level, '''View->Set Contour Parameters'''. In PolyScope uncheck '''Plane->Auto Contour Level''' and '''Strip->Auto Contour Level''', and use '''Plane->Set Contour Parameters''' and '''Strip->Set Contour Parameters'''. The last base contour level setting will be recalled the next time CARA is started. Spectra transformed with PROSA will usually be scaled so that RMS noise is 100, therefore the common base level for such spectra is 400. Spectra transformed with other software may need a different base level.

	Since 3D 13C-resolved NOESY, 3D (H)CCH-COSY and 3D (H)CCH-TOCSY are often recorded with folding in the '''Cinept''' dimension and 15N-resolved NOESY is folded in the 15N dimension, check the '''View->Show Folded''' box in all windows to allow proper navigation.		Since 3D 13C-resolved NOESY, 3D (H)CCH-COSY and 3D (H)CCH-TOCSY are often recorded with folding in the '''Cinept''' dimension and 15N-resolved NOESY is folded in the 15N dimension, check the '''View->Show Folded''' box in all windows to allow proper navigation.

	By default, spin systems in the '''(select residue)''' pull-down menu of all SystemScope windows are sorted according to spin system number. To sort them according to residue number, uncheck the '''View->Order by Strip''' box.		By default, spin systems in the '''(select residue)''' pull-down menu of all SystemScope windows are sorted according to spin system number. To sort them according to residue number, uncheck the '''View->Order by Strip''' box.

	In PolyScope windows 3 and 7 check the '''View->Show List''' to display a panel with a list of spin systems. Click on the '''Assig.''' column header to sort spin systems by residue number.		In PolyScope windows 3 and 7 check the '''View->Show List''' to display a panel with a list of spin systems. Click on the '''Assig.''' column header to sort spin systems by residue number.

	Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.		Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.