Measuring 15N T1 and T2 relaxation times (Bruker) - Revision history

Alex: moved Measuring 15N T1 and T2 Relaxation Times on Bruker Instruments to Measuring 15N T1 and T2 relaxation times (Bruker)

2009-12-16T21:36:33Z

moved Measuring 15N T1 and T2 Relaxation Times on Bruker Instruments to Measuring 15N T1 and T2 relaxation times (Bruker)

← Older revision	Revision as of 21:36, 16 December 2009
(No difference)

TheresaRamelot at 17:56, 16 November 2009

2009-11-16T17:56:19Z

← Older revision		Revision as of 17:56, 16 November 2009
Line 14:		Line 14:
	Typical [[Media:RelaxT1_122807.txt\|vdlist]] and [[Media:Relaxt2_122807.txt\|vclist]] files are attached.		Typical [[Media:RelaxT1_122807.txt\|vdlist]] and [[Media:Relaxt2_122807.txt\|vclist]] files are attached.

			<br>

			At Rutger's University, spectra are obtained in about 1hr on a 600 MHz Bruker TCI 1.7-mm MicroCyroprobe.  This probe provides S/N that is about one order of magnitute higher than a conventional 5-mm probe.<br>
	At Rutger's University, ~~we obtain the~~ spectra in about 1hr on a Bruker 1.7-mm ~~microcyroprobe~~.

	=== Processing 1D <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> on Bruker Spectrometers ===		=== Processing 1D <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> on Bruker Spectrometers ===

TheresaRamelot at 17:42, 16 November 2009

2009-11-16T17:42:59Z

← Older revision		Revision as of 17:42, 16 November 2009
Line 1:		Line 1:
	~~== '''Introduction''' ==~~

	The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ<sub>c</sub> >> 0.5 ns), the correlation time of a protein is related to the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times, and nuclear frequency (ν<sub>N</sub>) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.          [[Image:Tc eq1.png\|center\|250x50px]]

	In practice, global <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for an unknown protein target can be obtained quickly (ca. 1 h) on a 1.7-mm microcyroprobe using 1D <sup>15</sup>N-edited relaxation experiments (Ref. 2), by fitting integrated signal in the backbone amide <sup>1</sup>H region of the spectrum as a function of delay time to an exponential decay. One then computes the correlation time using Eq. 1, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size.  As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kiloDaltons. This approach is reliable up to MW ≈ 25 kDa, where accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes problematic.

	~~<br>~~

	== '''Methods''' ==		== '''Methods''' ==

TheresaRamelot at 17:40, 16 November 2009

2009-11-16T17:40:38Z

← Older revision		Revision as of 17:40, 16 November 2009
Line 21:		Line 21:

	Typical [[Media:RelaxT1_122807.txt\|vdlist]] and [[Media:Relaxt2_122807.txt\|vclist]] files are attached.		Typical [[Media:RelaxT1_122807.txt\|vdlist]] and [[Media:Relaxt2_122807.txt\|vclist]] files are attached.



			At Rutger's University, we obtain the spectra in about 1hr on a Bruker 1.7-mm microcyroprobe.

	=== Processing 1D <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> on Bruker Spectrometers ===		=== Processing 1D <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> on Bruker Spectrometers ===

Jma at 17:33, 11 November 2009

2009-11-11T17:33:38Z

← Older revision		Revision as of 17:33, 11 November 2009
Line 47:		Line 47:
	==== Figure 1:  Manual Integration of Amide Region in First Slice ====		==== Figure 1:  Manual Integration of Amide Region in First Slice ====

	[[Image:T1guide integration.png\|~~800x550px~~]]		[[Image:T1guide integration.png\|800px]]

	*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.		*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.
Line 53:		Line 53:
	==== Figure 2: Relaxation Window ====		==== Figure 2: Relaxation Window ====

	[[Image:T1guide relaxation.png\|~~800x550px~~]]		[[Image:T1guide relaxation.png\|800px]]

	*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.		*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.
Line 67:		Line 67:
	==== Figure 4:  Fitting the Data<br> ====		==== Figure 4:  Fitting the Data<br> ====

	[[Image:T1guide T1fit.png\|~~800x550px~~]]		[[Image:T1guide T1fit.png\|800px]]

	*Finally, the user can also display/print the report and print the plot.		*Finally, the user can also display/print the report and print the plot.
Line 79:		Line 79:
	==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====		==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====

	[[Image:T1wiki fig5.png\|~~800x600px~~]]<br>		[[Image:T1wiki fig5.png\|800px]]<br>

	== <br>'''References''' ==		== <br>'''References''' ==

Jma at 16:23, 11 November 2009

2009-11-11T16:23:38Z

← Older revision		Revision as of 16:23, 11 November 2009
Line 47:		Line 47:
	==== Figure 1:  Manual Integration of Amide Region in First Slice ====		==== Figure 1:  Manual Integration of Amide Region in First Slice ====

	[[Image:T1guide integration.png]]		[[Image:T1guide integration.png\|800x550px]]

	*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.		*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.
Line 53:		Line 53:
	==== Figure 2: Relaxation Window ====		==== Figure 2: Relaxation Window ====

	[[Image:T1guide relaxation.png]]		[[Image:T1guide relaxation.png\|800x550px]]

	*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.		*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.
Line 67:		Line 67:
	==== Figure 4:  Fitting the Data<br> ====		==== Figure 4:  Fitting the Data<br> ====

	[[Image:T1guide T1fit.png]]		[[Image:T1guide T1fit.png\|800x550px]]

	*Finally, the user can also display/print the report and print the plot.		*Finally, the user can also display/print the report and print the plot.
Line 79:		Line 79:
	==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====		==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====

	[[Image:~~T1wiki_fig5~~.png\|~~1200x800px~~]]<br>		[[Image:T1wiki fig5.png\|800x600px]]<br>

	== <br>'''References''' ==		== <br>'''References''' ==

Jma at 21:54, 10 November 2009

2009-11-10T21:54:41Z

← Older revision		Revision as of 21:54, 10 November 2009
Line 81:		Line 81:
	[[Image:T1wiki_fig5.png\|1200x800px]]<br>		[[Image:T1wiki_fig5.png\|1200x800px]]<br>

	== <br>'''~~REFERENCES~~''' ==		== <br>'''References''' ==

	1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay,L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003. <br>3.    Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. and Bax, A. (1992) Backbone dyanamics of calmodulin studied by <sup>15</sup>N relaxation using inverse detected two-dimensional NMR spectroscopy.~~ ~~ ''Biochemistry 31'', 5269-5278.<br><br>		1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay, L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003. <br>3.    Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. and Bax, A. (1992) Backbone dyanamics of calmodulin studied by <sup>15</sup>N relaxation using inverse detected two-dimensional NMR spectroscopy. ''Biochemistry 31'', 5269-5278.<br><br>

	-- JimAramini - 10 Nov 2009		-- JimAramini - 10 Nov 2009

Jma at 21:51, 10 November 2009

2009-11-10T21:51:58Z

← Older revision		Revision as of 21:51, 10 November 2009
Line 42:		Line 42:
	<pre>t1guide		<pre>t1guide
	</pre>		</pre>
	*Extract the first slice of the spectrum using the Extract Slice option and expand on the amide region.		*Extract the first slice of the spectrum using the Extract Slice option and expand on the amide region.
	*Proceed to Peaks/Ranges and perform a manual integration (Figure 2).  We typically integrate between δ ≈ 10 to 8.5 ppm.  Save the integration region to disk (the diskette/A symbol in the tool bar).<br>		*Proceed to Peaks/Ranges and perform a manual integration (Figure 2).  We typically integrate between δ ≈ 10 to 8.5 ppm.  Save the integration region to disk (the diskette/A symbol in the tool bar).<br>

	==== Figure 1:  Manual Integration of Amide Region in First Slice ====		==== Figure 1:  Manual Integration of Amide Region in First Slice ====

	[[Image:~~T1guide_integration~~.png]]		[[Image:T1guide integration.png]]

	*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.		*Proceed to the Relaxation Window.  A plot of integral area vs. delay time will be displayed (Figure 2).  Any points that are outliers can be deleted at this point using the mouse.

	==== Figure 2: Relaxation Window ====		==== Figure 2: Relaxation Window ====

	[[Image:~~T1guide_relaxation~~.png]]		[[Image:T1guide relaxation.png]]

	*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.		*Proceed to the Fitting Function (Figure 3).  Here the user inputs various parameters including the number of points and type of function used to fit the data. We typically use the uxnmrt1 function and we prefer to use the integral area.  Be sure to select the correct vdlist or vclist used in the experiment.

	==== Figure 3:  The Fitting Function ====		==== Figure 3:  The Fitting Function ====

	~~[[Image:T1guide_function.png]]~~

			[[Image:T1guide function.png]]

			<br>

	*Proceed to Start Calculation and fit the data (Figure 4)		*Proceed to Start Calculation and fit the data (Figure 4)

	==== Figure 4:  Fitting the Data<br> ====		==== Figure 4:  Fitting the Data<br> ====

	[[Image:~~T1guide_T1fit~~.png]]		[[Image:T1guide T1fit.png]]

	*Finally, the user can also display/print the report and print the plot.		*Finally, the user can also display/print the report and print the plot.

			<br>

	=== Compare the New τ<sub>c</sub> to a Standard Plot ===		=== Compare the New τ<sub>c</sub> to a Standard Plot ===

	The final step in the process is to compare your τ<sub>c</sub> to a standard plot of τ<sub>c</sub> (ns) vs. MW (kDa) for known monomers (Figure 5).  Temperature is an extremely important variable here, and it is recommended that all data be acquired at the same temperature, preferably on the same instrument.  At Rutgers we have a standard curve obtained at 298 K on our Bruker 600.  If your target is significantly above the curve, then it is not a monomer.		The final step in the process is to compare your τ<sub>c</sub> to a standard plot of τ<sub>c</sub> (ns) vs. MW (kDa) for known monomers (Figure 5).  Temperature is an extremely important variable here, and it is recommended that all data be acquired at the same temperature, preferably on the same instrument.  At Rutgers we have a standard curve obtained at 298 K on our Bruker 600.  If your target is significantly above the curve, then it is not a monomer.

	~~==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====~~


			==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====

	<br>		[[Image:T1wiki_fig5.png\|1200x800px]]<br>

	== <br>'''REFERENCES''' ==		== <br>'''REFERENCES''' ==

	1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay,L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003. <br>3.    Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. and Bax, A. (1992) Backbone dyanamics of calmodulin studied by <sup>15</sup>N relaxation using inverse detected two-dimensional NMR spectroscopy.  ''Biochemistry 31'', 5269-5278.<br><br>		1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay,L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003. <br>3.    Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. and Bax, A. (1992) Backbone dyanamics of calmodulin studied by <sup>15</sup>N relaxation using inverse detected two-dimensional NMR spectroscopy.  ''Biochemistry 31'', 5269-5278.<br><br>

	-- JimAramini - 10 Nov 2009		-- JimAramini - 10 Nov 2009

Jma at 21:41, 10 November 2009

2009-11-10T21:41:09Z

← Older revision		Revision as of 21:41, 10 November 2009
Line 73:		Line 73:


	=== Compare the New τ<sub>c</sub> to a Standard Plot ===		=== Compare the New τ<sub>c</sub> to a Standard Plot ===

	The final step in the process is to compare your τ<sub>c</sub> to a standard plot of τ<sub>c</sub> (ns) vs. MW (kDa) for known monomers (Figure 5).  Temperature is an extremely important variable here, and it is recommended that all data be acquired at the same temperature, preferably on the same instrument.  At Rutgers we have a standard curve obtained at 298 K on our Bruker 600.  If your target is significantly above the curve, then it is not a monomer.<br><br><br><br>'''REFERENCES'''~~<br><br>~~1.    Kay ~~et al~~. (1989, Biochemistry 28, 8972)<br>2.    Farrow ~~et al~~., 1994, Biochemistry 33, 5984<br>3.    Barbato ~~et al~~., Biochemistry 31, 5269-5278.<br><br>		The final step in the process is to compare your τ<sub>c</sub> to a standard plot of τ<sub>c</sub> (ns) vs. MW (kDa) for known monomers (Figure 5).  Temperature is an extremely important variable here, and it is recommended that all data be acquired at the same temperature, preferably on the same instrument.  At Rutgers we have a standard curve obtained at 298 K on our Bruker 600.  If your target is significantly above the curve, then it is not a monomer.

			==== Figure 5:  Plot of τ<sub>c</sub> vs. MW for Various NESG Monomers ====



			<br>

			== <br>'''REFERENCES''' ==

			1.    Kay, L.E., Torchia, D.A. and Bax, A. (1989) Backbone dynamics of proteins as studied by <sup>15</sup>N inverse detected heteronuclear NMR spectroscopy:  Application to staphylococcal nuclease. ''Biochemistry 28'', 8972-8979.<br>2.    Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M., Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D. and Kay,L.E. (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by <sup>15</sup>N NMR relaxation. ''Biochemistry 33'', 5984-6003. <br>3.    Barbato, G., Ikura, M., Kay, L.E., Pastor, R.W. and Bax, A. (1992) Backbone dyanamics of calmodulin studied by <sup>15</sup>N relaxation using inverse detected two-dimensional NMR spectroscopy.  ''Biochemistry 31'', 5269-5278.<br><br>

	-- JimAramini - 10 Nov 2009		-- JimAramini - 10 Nov 2009

Jma at 21:30, 10 November 2009

2009-11-10T21:30:14Z

@@ Line 1: / Line 1: @@
 == '''Introduction'''  ==
-The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ<sub>c</sub> &gt;&gt; 0.5 ns), the correlation time of a protein is related to the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times, and nuclear frequency (ν<sub>N</sub>) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; [[Image:Tc eq1.png|center|300x60px]]
+The rotational correlation time of a protein in solution is the time for a protein to rotate one radian. In the limit of slow molecular motion (τ<sub>c</sub> &gt;&gt; 0.5 ns), the correlation time of a protein is related to the ratio of the longitudinal (T<sub>1</sub>) and transverse (T<sub>2</sub>) <sup>15</sup>N relaxation times, and nuclear frequency (ν<sub>N</sub>) according to Eq. 1, which is derived from Eq. 8 in Kay et al. (Ref. 1) by considering only J(0) and J(ω) spectral densities and neglecting higher frequency terms.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; [[Image:Tc eq1.png|center|250x50px]]
 In practice, global <sup>15</sup>N T<sub>1</sub> and T<sub>2</sub> relaxation times for an unknown protein target can be obtained quickly (ca. 1 h) on a 1.7-mm microcyroprobe using 1D <sup>15</sup>N-edited relaxation experiments (Ref. 2), by fitting integrated signal in the backbone amide <sup>1</sup>H region of the spectrum as a function of delay time to an exponential decay. One then computes the correlation time using Eq. 1, and compares it to a standard curve of τ<sub>c</sub> vs. protein molecular weight (MW) obtained at the same temperature on a series of known monomeric proteins of varying size.&nbsp; As a general rule of thumb, the τ<sub>c</sub> of a monomeric protein in solution in nanoseconds is approximately 0.6 times its molecular weight in kiloDaltons. This approach is reliable up to MW ≈ 25 kDa, where accurate measurement of the diminishing <sup>15</sup>N T<sub>2</sub> becomes problematic.
@@ Line 20: / Line 20: @@
 *set d1 to 3 - 5 s (T<sub>1</sub>) or 1.5 - 3 s (T<sub>2</sub>) for adequate relaxation during the experiment.
-Typical [[Media:RelaxT1_122807.txt|vdlist]] and [[Media:Relaxt2_122807.txt|vclist]] files are attached.<br><br>
+Typical [[Media:RelaxT1_122807.txt|vdlist]] and [[Media:Relaxt2_122807.txt|vclist]] files are attached.