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

Alex: moved Estimation of rotational correlation time to Measuring 15N T1 and T2 relaxation times (Varian)

2009-12-16T21:36:49Z

moved Estimation of rotational correlation time to Measuring 15N T1 and T2 relaxation times (Varian)

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

Alex at 21:35, 16 December 2009

2009-12-16T21:35:06Z

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Aringh at 15:41, 15 December 2009

2009-12-15T15:41:44Z

← Older revision		Revision as of 15:41, 15 December 2009
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	== '''Correlation Time and NMR Measurement Time Prediction''' ==		== '''Correlation Time and NMR Measurement Time Prediction''' ==

	=== '''Experimental Setup''' ===		=== '''Experimental Setup''' ===

	Run the <tt>HTP_t1t2_setup</tt> script to setup the following experiments:		Run the <tt>HTP_t1t2_setup</tt> script to setup the following experiments:
	* 2D [15N, 1H] HSQC, ~1.5h for SN analysis
	** <tt>NHonly='n' C13refoc='y' ni=256 f1180='n' phase=1,2 nt=8 ss=32</tt>
	* 1D 15N T1, ~30 min
	** <tt>ni=1 phase=1 T1='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128</tt>
	** <tt>relaxT=0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.5, 2.0</tt>
	* 1D 15N T2, ~30 min
	** <tt>ni=1 phase=1 T2='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128</tt>
	** <tt>relaxT=0.01,0.03,0.05,0.07,0.09,0.11,0.13,0.15,0.17 maxrelaxT=0.17</tt>

			*2D [15N, 1H] HSQC, ~1.5h for SN analysis
			**<tt>NHonly='n' C13refoc='y' ni=256 f1180='n' phase=1,2 nt=8 ss=32</tt>
			*1D 15N T1, ~30 min
			**<tt>ni=1 phase=1 T1='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128</tt>
			**<tt>relaxT=0.1, 0.2, 0.3, 0.4, 0.7, 1.0, 1.5, 2.0</tt>
			*1D 15N T2, ~30 min
			**<tt>ni=1 phase=1 T2='y' f1180='n' NHonly='n' C13refoc='n' ss=256 nt=128</tt>
			**<tt>relaxT=0.01,0.03,0.05,0.07,0.09,0.11,0.13,0.15,0.17 maxrelaxT=0.17</tt>

	Key issues:		<br> Key issues:
	* For most proteins it is recommended to run T1 and T2 experiments for at least 30 min each to achieve adequate S/N on a room-temperature probe. Short measurement times may lead to underestimated tc values. With cryogenic probes or very concentrated (> 1 mM) samples the minimum measurement time may be smaller.
	* Short <tt>d1</tt> delays (~1 s) may lead to incorrect integral for the first T1 point. However, long d1 values may lead to a large residual water line and non-uniform baseline, especially on a cryoprobe. See what works best for the particular sample and spectrometer.
	* <tt>relaxT</tt> must be given as a multiple of 10 ms for T1 and '''odd''' multiple of 10 ms for T2.
	* <tt>maxrelaxT</tt> is not used in T1 measurements.
	* Avoid sampling T2 points beyond 250 ms - it may cause excessive sample heating.
	* Intermediate tc values (between monomer and dimer) may indicate transient dimerization. Dilution studies are then required.
	* The tc value is calculated under assumption of isotropic tumbling. For example, if a protein consists of long parallel a-helices the reported tc will indicate a larger molecular weight.

	~~===~~ '''~~Correlation Time Measurement~~''' ~~===~~		*For most proteins it is recommended to run T1 and T2 experiments for at least 30 min each to achieve adequate S/N on a room-temperature probe. Short measurement times may lead to underestimated tc values. With cryogenic probes or very concentrated (> 1 mM) samples the minimum measurement time may be smaller.
			*Short <tt>d1</tt> delays (~1 s) may lead to incorrect integral for the first T1 point. However, long d1 values may lead to a large residual water line and non-uniform baseline, especially on a cryoprobe. See what works best for the particular sample and spectrometer.
			*<tt>relaxT</tt> must be given as a multiple of 10 ms for T1 and '''odd''' multiple of 10 ms for T2.
			*<tt>maxrelaxT</tt> is not used in T1 measurements.
			*Avoid sampling T2 points beyond 250 ms - it may cause excessive sample heating.
			*Intermediate tc values (between monomer and dimer) may indicate transient dimerization. Dilution studies are then required.
			*The tc value is calculated under assumption of isotropic tumbling. For example, if a protein consists of long parallel a-helices the reported tc will indicate a larger molecular weight.

	~~Based on the Stokes~~'s law, the isotropic rotational correlation time for approximately spherical globular proteins is a function of the effective hydrodynamic radius of the protein, which provides a simple way to check the oligomerizaiton status of protein in solution. Here is the basic law: the correlation time in nanoseconds is approximately half of the value of the protein'~~s molecular weight in kilodaltons.~~		=== '''Correlation Time Measurement''' ===

	~~A protein~~'s rotational correlation time ~~(Tauc) can be quickly estimated from average 15N T1 and T2 relaxation times~~.		Based on the Stokes's law, the isotropic rotational correlation time for approximately spherical globular proteins is a function of the effective hydrodynamic radius of the protein, which provides a simple way to check the oligomerizaiton status of protein in solution. Here is the basic law: the correlation time in nanoseconds is approximately half of the value of the protein's molecular weight in kilodaltons.

			A protein's rotational correlation time (Tauc) can be quickly estimated from average 15N T1 and T2 relaxation times.

	To calculate Tauc:		<br> To calculate Tauc:
	* Process the data in Buffalo.VNMR with <tt>wft</tt> and adjust the phase for pure absorption.
	* Invoke <tt>dc</tt> to correct baseline shift and slope. You can expand the spectrum for better results.
	* Invoke <tt>dscale</tt> and <tt>setref</tt> to make sure the ppm scale is correct - required for the <tt>tc</tt> macro.
	* Display all spectra with <tt>dssh</tt> and check that
	** baseline is flat and uniform across points.
	** Spectral intensity follows an exponential decay.
	* Run <tt>tc([T1exp, T2exp, [ppm1, ppm2]])</tt>, where the first two arguments are T1 and T2 experiment numbers, and the last two optional arguments specify the integration range in ppm. By default, integration is performed over the range between 10.5 ppm and 8.5 ppm to exclude signals from side-chain CONH2 groups (6 - 7 ppm) and unfolded parts (8 ppm).

			*Process the data in Buffalo.VNMR with <tt>wft</tt> and adjust the phase for pure absorption.
			*Invoke <tt>dc</tt> to correct baseline shift and slope. You can expand the spectrum for better results.
			*Invoke <tt>dscale</tt> and <tt>setref</tt> to make sure the ppm scale is correct - required for the <tt>tc</tt> macro.
			*Display all spectra with <tt>dssh</tt> and check that
			**baseline is flat and uniform across points.
			**Spectral intensity follows an exponential decay.
			*Run <tt>tc([T1exp, T2exp, [ppm1, ppm2]])</tt>, where the first two arguments are T1 and T2 experiment numbers, and the last two optional arguments specify the integration range in ppm. By default, integration is performed over the range between 10.5 ppm and 8.5 ppm to exclude signals from side-chain CONH2 groups (6 - 7 ppm) and unfolded parts (8 ppm).

			<br>

	==== '''Tc macro''' ====		==== '''Tc macro''' ====
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	#Unpack it with <tt>tar xvf tc_macro.tar</tt>		#Unpack it with <tt>tar xvf tc_macro.tar</tt>

	=== '''Measurement Time Prediction''' ===		=== '''Measurement Time Prediction''' ===

	The minimum measurement time of (4,3)D GFT experiments can be reliably predicted from the S/N distribution of a 2D [15N, 1H] HSQC and the rotational correlation time tau_c. First, you need to generate an integrated peaklist.		The minimum measurement time of (4,3)D GFT experiments can be reliably predicted from the S/N distribution of a 2D [15N, 1H] HSQC and the rotational correlation time tau_c. First, you need to generate an integrated peaklist.

	==== '''Peak Integration in 2D [15N, 1H] HSQC''' ====		==== '''Peak Integration in 2D [15N, 1H] HSQC''' ====

	Having processed 2D (15N, 1H) HSQC as described in data process, one can do the peak picking and integration of 2D (15N, 1H) HSQC ~~manualy~~ or semi-~~automaticlly~~ as following:		Having processed 2D (15N, 1H) HSQC as described in data process, one can do the peak picking and integration of 2D (15N, 1H) HSQC manually or semi-automatically as following:

	*Peak picking and integration by using program XEASY		*Peak picking and integration by using program XEASY
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	##Use <tt>in</tt> to automatic pick peaks with total peak number slightly more than expected by select adjust contour level; then manually remove side-chain amide peaks		##Use <tt>in</tt> to automatic pick peaks with total peak number slightly more than expected by select adjust contour level; then manually remove side-chain amide peaks
	##Use <tt>mn</tt> to measure noise level, and use <tt>tw</tt> to display the noise level value. Normally the value of 2.5 times of standard deviation ( ~250 if the noise level has been normalized ) is taken as noise level.		##Use <tt>mn</tt> to measure noise level, and use <tt>tw</tt> to display the noise level value. Normally the value of 2.5 times of standard deviation ( ~250 if the noise level has been normalized ) is taken as noise level.
	##Use <tt>ip</tt> to choose peak height (m) as integration mode, and use "ii" to integrate the whole spectrum. An [[NESG:XEASY\|XEASY]] external program "PeakintI" can be used to obtain more accurate peak height values, which is ~~discribed~~ below.		##Use <tt>ip</tt> to choose peak height (m) as integration mode, and use "ii" to integrate the whole spectrum. An [[NESG:XEASY\|XEASY]] external program "PeakintI" can be used to obtain more accurate peak height values, which is described below.
	##Use <tt>wp</tt> to save the peak list.		##Use <tt>wp</tt> to save the peak list.

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	The SN distribution of resonances in a NMR spectra can be fit to the Gaussian distribution:		The SN distribution of resonances in a NMR spectra can be fit to the Gaussian distribution:

	[[Image:~~Sn_nhsqc~~.jpg]]		[[Image:Sn nhsqc.jpg]]

	<br><tt>f= aexp(-0.5((ln(SN_i)-ln(SN_0))/b)^2) </tt><br> where <tt>SN_0</tt> is the most populated S/N observed, <tt>f is the expected population at a certern SN value SN_i</tt>, <tt>a and b</tt> are contants.		<br><tt>f= aexp(-0.5((ln(SN_i)-ln(SN_0))/b)^2) </tt><br> where <tt>SN_0</tt> is the most populated S/N observed, <tt>f is the expected population at a certern SN value SN_i</tt>, <tt>a and b</tt> are contants.

Alex: moved NESG:TcAndPredictionOfMeasurementTime to Estimation of rotational correlation time

2009-11-10T23:59:22Z

moved NESG:TcAndPredictionOfMeasurementTime to Estimation of rotational correlation time

← Older revision	Revision as of 23:59, 10 November 2009
(No difference)

Admin at 16:57, 20 October 2009

2009-10-20T16:57:15Z

← Older revision		Revision as of 16:57, 20 October 2009
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	Assuming that a peak shall have at least SN value of 2 in order to be observed or detected, and the average b for (4,3) GFT experiments is 0.8; if 95% peak detection yield is required, the exptected SN_0 is:<br><br><tt>SN_0=exp(ln2+1.644*b)=7.4 </tt><br><br>		Assuming that a peak shall have at least SN value of 2 in order to be observed or detected, and the average b for (4,3) GFT experiments is 0.8; if 95% peak detection yield is required, the exptected SN_0 is:<br><br><tt>SN_0=exp(ln2+1.644*b)=7.4 </tt><br><br>

	~~<img src="%ATTACHURLPATH%/sn2~~.jpg~~" alt="sn2.jpg" width='625' height='328' />~~		[[Image:Sn2.jpg]]

	NMR measurement time of (4,3) GFT HNNCABCA, (4,3)D GFT CABCAcoNHN and (4,3)D HABCABCONHN can be calculated from the following equation:<br><br> <tt>T_43d= ((SN_43d* Tauc^2)/(SN_2d*A))^2 </tt><br> where <tt>T_43d</tt> is the required time for (4,3) GFT experiment, <tt>SN_43d</tt> is the expected SN value of (4,3)D GFT experiments, <tt>SN_2d</tt> is the SN per hour of 2D (15N, 1H) HSQC. =A= is constant, which has value of 0.8639 for (4,3) GFT HNNCABCA, 1.6019 for (4,3)D GFT CABCAcoNHN and 1.0153 for (4,3)D HABCABCONHN.		NMR measurement time of (4,3) GFT HNNCABCA, (4,3)D GFT CABCAcoNHN and (4,3)D HABCABCONHN can be calculated from the following equation:<br><br> <tt>T_43d= ((SN_43d* Tauc^2)/(SN_2d*A))^2 </tt><br> where <tt>T_43d</tt> is the required time for (4,3) GFT experiment, <tt>SN_43d</tt> is the expected SN value of (4,3)D GFT experiments, <tt>SN_2d</tt> is the SN per hour of 2D (15N, 1H) HSQC. =A= is constant, which has value of 0.8639 for (4,3) GFT HNNCABCA, 1.6019 for (4,3)D GFT CABCAcoNHN and 1.0153 for (4,3)D HABCABCONHN.

	~~<img src="%ATTACHURLPATH%/sn3~~.jpg~~" alt="sn3.jpg" width='834' height='362' />~~		[[Image:Sn3.jpg]]

	One can use [[NESG:UBNMR\|UBNMR]] to run the measurement time predition by the following command: <br><br><br> <tt>predict T2d SN2d Tc</tt><br>where		One can use [[NESG:UBNMR\|UBNMR]] to run the measurement time predition by the following command: <br><br><br> <tt>predict T2d SN2d Tc</tt><br>where

Admin at 16:53, 20 October 2009

2009-10-20T16:53:02Z

← Older revision		Revision as of 16:53, 20 October 2009
Line 85:		Line 85:
	The SN distribution of resonances in a NMR spectra can be fit to the Gaussian distribution:		The SN distribution of resonances in a NMR spectra can be fit to the Gaussian distribution:

	~~<img src="%ATTACHURLPATH%/sn_nhsqc~~.jpg~~" alt="sn_nhsqc.jpg"/>~~		[[Image:Sn_nhsqc.jpg]]

	<br><tt>f= aexp(-0.5((ln(SN_i)-ln(SN_0))/b)^2) </tt><br> where <tt>SN_0</tt> is the most populated S/N observed, <tt>f is the expected population at a certern SN value SN_i</tt>, <tt>a and b</tt> are contants.		<br><tt>f= aexp(-0.5((ln(SN_i)-ln(SN_0))/b)^2) </tt><br> where <tt>SN_0</tt> is the most populated S/N observed, <tt>f is the expected population at a certern SN value SN_i</tt>, <tt>a and b</tt> are contants.

Admin at 16:34, 20 October 2009

2009-10-20T16:34:56Z

← Older revision		Revision as of 16:34, 20 October 2009
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	##Use <tt>wp</tt> to save the peak list.		##Use <tt>wp</tt> to save the peak list.

	*More accurate peak height measurement by using program [[NESG:~~Peakintl~~\|PeakintI]].<br> Type command: <br><br> <tt>peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.		*More accurate peak height measurement by using program [[NESG:PeakintI\|PeakintI]].<br> Type command: <br><br> <tt>peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.
	*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>		*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>
	**by using [[NESG:UBNMR\|UBNMR]], please check [[NESG:UBNMR\|UBNMR]] macro		**by using [[NESG:UBNMR\|UBNMR]], please check [[NESG:UBNMR\|UBNMR]] macro

Admin at 16:29, 20 October 2009

2009-10-20T16:29:42Z

← Older revision		Revision as of 16:29, 20 October 2009
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	##Use <tt>in</tt> to automatic pick peaks with total peak number slightly more than expected by select adjust contour level; then manually remove side-chain amide peaks		##Use <tt>in</tt> to automatic pick peaks with total peak number slightly more than expected by select adjust contour level; then manually remove side-chain amide peaks
	##Use <tt>mn</tt> to measure noise level, and use <tt>tw</tt> to display the noise level value. Normally the value of 2.5 times of standard deviation ( ~250 if the noise level has been normalized ) is taken as noise level.		##Use <tt>mn</tt> to measure noise level, and use <tt>tw</tt> to display the noise level value. Normally the value of 2.5 times of standard deviation ( ~250 if the noise level has been normalized ) is taken as noise level.
	##Use <tt>ip</tt> to choose peak height (m) as integration mode, and use "ii" to integrate the whole spectrum. An XEASY external program "PeakintI" can be used to obtain more accurate peak height values, which is discribed below.		##Use <tt>ip</tt> to choose peak height (m) as integration mode, and use "ii" to integrate the whole spectrum. An [[NESG:XEASY\|XEASY]] external program "PeakintI" can be used to obtain more accurate peak height values, which is discribed below.
	##Use <tt>wp</tt> to save the peak list.		##Use <tt>wp</tt> to save the peak list.

	*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br><br> <tt>peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.		*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br><br> <tt>peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.
	*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>		*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>
	**by using UBNMR, please check UBNMR macro		**by using [[NESG:UBNMR\|UBNMR]], please check [[NESG:UBNMR\|UBNMR]] macro
	**by using script '''sim''', run macro '''comb_yang''' by typing <tt>sim comb_yang</tt>		**by using script '''sim''', run macro '''comb_yang''' by typing <tt>sim comb_yang</tt>

Admin at 16:28, 20 October 2009

2009-10-20T16:28:44Z

← Older revision		Revision as of 16:28, 20 October 2009
Line 76:		Line 76:
	##Use <tt>wp</tt> to save the peak list.		##Use <tt>wp</tt> to save the peak list.

	*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br> <tt> peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.		*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br><br> <tt>peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.
	*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>		*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>
	**by using UBNMR, please check UBNMR macro		**by using UBNMR, please check UBNMR macro

Admin at 16:28, 20 October 2009

2009-10-20T16:28:15Z

← Older revision		Revision as of 16:28, 20 October 2009
Line 76:		Line 76:
	##Use <tt>wp</tt> to save the peak list.		##Use <tt>wp</tt> to save the peak list.

	*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br> <~~nowiki~~> peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </~~nowiki~~><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.		*More accurate peak height measurement by using program [[NESG:Peakintl\|PeakintI]].<br> Type command: <br> <tt> peakintI ../data/NHsqc6001 nhsqcsa_b.peaks 250 -i -t 2 2 0.1 </tt><br> where the <tt>nhsqcsa_b.peaks</tt> is the input peak list and the output file will be <tt>inhsqcsa_b.peaks</tt>.
	*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>		*Combing atom name informaiton in the peak list for S/N distribution (optional). <br>
	**by using UBNMR, please check UBNMR macro		**by using UBNMR, please check UBNMR macro