NESG Wiki - User contributions [en]

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-11-03T16:56:46Z

Agutmana: /* Pulse program modifications */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request (see [[Setting up non-uniformly sampled spectra]]):
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer (NB! The directory may be slightly different on your system).<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-11-03T16:55:53Z

Agutmana: /* Pulse program modifications */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request [[Setting up non-uniformly sampled spectra]]:
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer (NB! The directory may be slightly different on your system).<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-11-03T16:53:49Z

Agutmana: /* Pulse program modifications */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request []:
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer (NB! The directory may be slightly different on your system).<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:54:39Z

Agutmana: /* Software requests */

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by [http://www.nmr.gu.se/english/research/orekhov Vladislav Orekhov] (Swedish NMR Centre, University of Gothenburg). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group or by e-mailing (gutmanas@ebi.ac.uk)
*Mddnmr software is developed by [http://www.nmr.gu.se/english/research/orekhov Vladislav Orekhov] (Swedish NMR Centre, University of Gothenburg). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:52:16Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by [http://www.nmr.gu.se/english/research/orekhov Vladislav Orekhov] (Swedish NMR Centre, University of Gothenburg). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:49:35Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([http://www.nmr.gu.se/english Swedish NMR Centre, University of Gothenburg]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:48:10Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([http://www.nmr.gu.se | Swedish NMR Centre, University of Gothenburg]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:47:43Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([http://www.nmr.gu.se|Swedish NMR Centre, University of Gothenburg]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:47:23Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([[http://www.nmr.gu.se|Swedish NMR Centre, University of Gothenburg]]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:46:55Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([[Swedish NMR Centre, University of Gothenburg|http://www.nmr.gu.se]]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:46:28Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov ([Swedish NMR Centre, University of Gothenburg|http://www.nmr.gu.se]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:45:52Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, [http://www.nmr.gu.se]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Processing non-uniformly sampled spectra with Multidimensional Decomposition

2010-07-26T12:42:12Z

Agutmana:

__NORICHEDITOR__
== Introduction and Prerequisites ==

This section gives a step-by-step manual to processing with the MDDGUI program, developed in the Arrowsmith lab (University Health Network and University of Toronto). It is designed for processing 3D spectra collected with non-uniform or non-linear sampling (NUS). The spectra can be collected either on Varian spectrometers with later versions of BioPack installed ([[Setting up non-uniformly sampled spectra/NUS guide for Varian|click here for the relevant guide for Varian spectrometers]]) or on Bruker spectrometers running TopSpin ([[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|click here for the Arrowsmith protocol for NUS data collection on Bruker instruments]]).

The GUI package requires the '''mddnmr''' software developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, [[http://www.nmr.gu.se]]). Please contact him for your copy of the software. The GUI can be requested from the Arrowsmith group. You need Qt libraries in order to compile the GUI, or you can use the precompiled Linux version. Please refer to the installation README file for details. In addition to these two packages, you should have working nmrPipe and nmrDraw programs, as apodization, Fourier transforms, etc are performed by nmrPipe.

__NORICHEDITOR__

== Processing with MDDGUI ==
__NORICHEDITOR__

Before the Multi-dimensional decomposition can be started, the raw data has to be converted through a number of steps to an appropriate format. The steps below are accompanied by the GUI screen shots. To move between the different stages of the processing, use "Next" and "Back" buttons. Refer to README file or tutorial for more specific instructions.

<br>
__NORICHEDITOR__
=== Step 1. Reshuffle FIDs ===
__NORICHEDITOR__

Rearrange the FIDs in the '''ser''' of '''fid''' file so that they are in correct positions for an imaginary case as if the data set was sampled regularly. The missing FIDs are replaced by zeros as dictated by the vdlist or procpar files. First, click the appropriate radio button, edit any file names if necessary, and click '''Insert Zeros''' before proceeding. <br>[[Image:MDDGUIstep1.jpg|thumb|left|505px|Figure 1a. Reshuffling FIDs in Bruker <tt>ser</tt> file.]][[Image:MDDGUIstep1 Varian.jpg|thumb|right|505px|Figure 1b. Reshuffling FIDs in Varian <tt>fid</tt> file.]]
<br style="clear: both" />

<br> <br>
__NORICHEDITOR__
=== Step 2. Convert to NMRPipe format ===
__NORICHEDITOR__

In this step there are a few parameters that the GUI might read wrong, especially with later versions of TopSpin. You can edit it as a simple bruk2pipe or var2pipe script. Pay extra attention to carrier and spectrometer frequencies, matrix sizes and acquisition modes (highlighted). As far as indirect dimensions' acquisition modes are concerned, it is important to distinguish between Echo-AntiEcho (Rance-Kay) and Complex/States-TPPI modes, as one can select within the group by adjusting flags for "nmrPipe -fn FT" command. In TopSpin2.1 we discovered that sweep width is sometimes not recorded in the acqu* files, therefore check that as well. Click '''Save''', then '''Run'''. <br>

<br>

<br>[[Image:MDDGUIstep2.jpg|thumb|left|505px|Figure 2a. Making sure the highlighted parameters are correct]][[Image:MDDGUIstep2 out.jpg|thumb|right|505px|Figure 2b. Output of the conversion to nmrPipe format]] NB! Make sure the spectrometer input is the reshuffled file (ser.sp or fid.sp)! <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 3. Process the first plane <br> ===
__NORICHEDITOR__

For the Bruker data, the first plane is regularly sampled while for Varian it is  not, which may make this step more difficult for Varian data. One only needs to adjust phasing, apodisation, region of interest and other parameters for the '''acquisition '''or directly detected dimension. The better the phasing, the better the convergence and the fewer the artefacts. It will be difficult to change the acquisition dimension parameters afterwards without redoing the calculation, so spend a few rounds with NMRDraw until you are happy and the phases are set properly (see highlighted section in the figure). As usual, click "Save", then "Run" each time you want to reprocess the plane. Button "nmrDraw" simply calls that program.

<br> [[Image:MDDGUIstep3.jpg|thumb|left|505px|Figure 3a. Initial script to process the first plane of the spectrum.]][[Image:MDDGUIstep3 final.jpg|thumb|right|505px|Figure 3b. After the adjustment of relevant parameters.]] [[Image:MDDGUIstep3 plane.jpg|thumb|center|1050px|Figure 3c. NMRDraw screenshot for phasing the acquisition dimension.]] <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 4. Processing the acquisition dimension<br> ===
__NORICHEDITOR__

Using the parameters from step 3, Fourier transform (FT) the acquisition dimension only for the whole data set and extract the region of interest. Note that the parameters are copied, but one can still edit them before proceeding. <br> [[Image:MDDGUIstep4.jpg|thumb|center|505px| Figure 4. Processing the acquisition dimension.]]<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 5. Converting to MDDNMR format<br> ===
__NORICHEDITOR__

Divide the the partially Fourier transformeded but still sparse spectrum into a number of regions overlapping by their acquisition dimension and convert these regions to MDDNMR format. This is the step before proceeding to calculations themselves. Make sure you select "Multiregion" radiobutton. Parallel computation is becoming obsolete, although is supported for now. Region width should be given in ppm

<br> [[Image:MDDGUIstep5.jpg|thumb|left|505px|Figure 5a. Slicing the spectrum into overlapping regions and converting to MDDNMR format.]][[Image:MDDGUIstep5 out.jpg|thumb|right|505px|Figure 5b. Output of the conversion to MDDNMR format.]] <br> <br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 6. Run MDD <br> ===
__NORICHEDITOR__

As simple as that. Check README for more detailed instructions. The most important parameter is the number of components (per region), which should be the maximum number of HSQC/projection peaks . You might need to do some editing of the "runmdd.sh" script depending on cluster availability (''e.g.'', you may need to remove & from the mddnmr command if your calculation is running on a desktop). In the Arrowsmith group, we usually open a separate terminal window, login to our cluster and start the calculations from there. '''Do not close the GUI''' in the meantime. If you do, it is a bit tricky to restart from where you are.

<br> [[Image:MDDGUIstep6.jpg|thumb|left|505px|Figure 6a. Parameters for the MDDNMR run.]] [[Image:MDDGUIstep6 run.jpg|thumb|right|505px|Figure 6b. How to start the calculation on a cluster.]]
<br style="clear: both" />

__NORICHEDITOR__
=== Step 7. Reconstructing the spectrum ===
__NORICHEDITOR__

Using the output of MDD, replace the zeros ( ''i.e.'' missing FIDs) by the reconstructed values. Here, if you had the GUI open through the previous step, the values will be filled automatically. If you did close it, restart it, go to step 5, choose "Multiregion" radiobutton, proceed to step 7 and fill out the values. That was the tricky part.

<br> [[Image:MDDGUIstep7.jpg|thumb|left|505px|Figure 7a. Reconstructing the full spectrum.]] [[Image:MDDGUIstep7 out.jpg|thumb|right|505px|Figure 7b. Output messages from the reconstruction step.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
=== Step 8. Fourier transform of indirect dimensions ===
__NORICHEDITOR__

This is the final step of the processing. Here the task is purely NMRPipe. You may do to your indirect dimensions what you need to do - apodization, baseline correction, etc, etc. You may need to play around with "FT -auto" if your peaks are not making sense. That is mainly because the GUI can't easily distinguish between "Complex", "States-TPPI" and related modes. So you may need to change the FT flags to rectify the situation.

{| border="1"
|+ '''Correspondence of FT flags to Complex acquisition modes'''
|-
! FT flag(s)
! Aquisition mode
! Action(s) performed
! Effect(s) on spectrum
|-
| FT
| Complex
| none
| none
|-
| FT -alt
| States-TPPI
| sign alternation for every other point
| Left and right (top and bottom) halves swapped
|-
| FT -neg
| Complex-N
| negation of imaginaries
| spectrum inverted
|-
| FT -alt -neg
| States-TPPI-N
| sign alternation and negation of imaginaries
| spectrum halves swapped and the whole spectrum inverted
|}

Finally, you don't need the GUI anymore, as you can edit the script to your liking with any text editor.

<br> [[Image:MDDGUIstep8.jpg|thumb|center|505px|Figure 8. Finalizing the Fourier transform of the indirect dimensions.]]

<br>
<br style="clear: both" />

__NORICHEDITOR__
== Cleaning up ==

Once you have processed your spectrum, there are a lot of temporary files that are no longer needed. By deleting them you can sometimes free up to a Gb of disk space. If you leave the standard names for these files you can delete the following:

{|
|-
! Bruker
! Varian
|-
| <pre> > rm -rf data/ pdata/ mdd_out_* ser.sp ft/test_nus1.dat
> gzip ser</pre>
| <pre> > rm -rf data/ mdd_out_* fid.sp ft/test_nus1.dat
> gzip fid </pre>
|}

You should, of course, modify accordingly, if your file names are different. In this particular example, one could also delete the ft/aliNoesy.ft1 file. Note also, that if you perform the processing on the spectrometer, it is desirable to keep the pdata/ directory intact.

<br>

== Software requests ==

<br>

*MDDGUI package can be requested from the Arrowsmith group (agutmana@uhnres.utoronto.ca)
*Mddnmr software is developed by Vladislav Orekhov (Swedish NMR Centre, University of Gothenburg, http://www.nmr.gu.se/~mdd). Please contact him for your copy of the package. <br>

Setting up non-uniformly sampled spectra

2010-07-26T12:39:52Z

Agutmana:

This section describes the acquisition of non-uniformly sampled (NUS) 3D data tailored to subsequent processing with Multidimensional Decomposition (MDD) software, described elsewhere on this site ([[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Click here to read about the processing with MDD]]). The intention of recoding NUS data is to either speed up the acquisition for high sensitivity experiments and/or concentrated samples, or to increase resolution for other cases.  The package is available for academic users. Please contact Aleks Gutmanas (gutmanas@ebi.ac.uk) to obtain your copy.

[[Setting up non-uniformly sampled spectra/NUS guide for Varian|Setting up NUS spectra on Varian spectrometers with BioPack]]

[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|Setting up NUS spectra on Bruker spectrometers]] according to the practice in the Arrowsmith group (University Health Network and University of Toronto)

Setting up non-uniformly sampled spectra

2010-07-26T12:39:22Z

Agutmana:

This section describes the acquisition of non-uniformly sampled (NUS) 3D data tailored to subsequent processing with Multidimensional Decomposition (MDD) software, described elsewhere on this site ([[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Click here to read about the processing with MDD]]). The intention of recoding NUS data is to either speed up the acquisition for high sensitivity experiments and/or concentrated samples, or to increase resolution for other cases.  The package is available for academic users. Please contact Aleks Gutmanas ([[Mailto:gutmanas@ebi.ac.uk]]) to obtain your copy.

[[Setting up non-uniformly sampled spectra/NUS guide for Varian|Setting up NUS spectra on Varian spectrometers with BioPack]]

[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|Setting up NUS spectra on Bruker spectrometers]] according to the practice in the Arrowsmith group (University Health Network and University of Toronto)

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-01-07T17:11:56Z

Agutmana: /* Special Remarks */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request from the Arrowsmith group (agutmana@uhnres.utoronto.ca):
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer (NB! The directory may be slightly different on your system).<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-01-07T17:11:19Z

Agutmana: /* Generating the sampling schedule and running the experiment */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request from the Arrowsmith group (agutmana@uhnres.utoronto.ca):
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer (NB! The directory may be slightly different on your system).<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features, worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto

2010-01-07T17:10:21Z

Agutmana: /* Generating the sampling schedule and running the experiment */

__NORICHEDITOR__
__TOC__
== Setting up acquisition of non-uniformly sampled 3D spectra on Bruker spectrometers with TopSpin ==
__NORICHEDITOR__

A standardized way to set non-uniform sampling (NUS) for TopSpin is in the making, however, while it is not yet included in the current distribution, this page will focus on the way Non-uniform sampling of 3D spectra is implemented by the Arrowsmith group in Toronto (University Health Network and University of Toronto). It is tailored for the subsequent processing with MDDGUI software, described elsewhere on this site.<br>

To set up 3D NUS experiments according to this method one needs the following:<br>

*Modified pulse program(s)

*A Jython script for generating the sampling schedule<br>

__NORICHEDITOR__
=== Pulse program modifications<br> ===
__NORICHEDITOR__

First of all, one has to set up a regular experiment with all pulses and delays calibrated as usual. Once this step is finished, one needs to replace the pulse program by a modified one.

This section briefly describes the essential pulse sequence modifications. The <sup>15</sup>N-NOESY-HSQC pulse program is used as an example. In most cases one just need to copy these changes into relevant pulse program.<br>

*Additional definitions should come with other definitions in the file:<br>
<pre>;********Added for NUS*******
define delay short
"short=50u"
"l2=td2/2"
"l3=td1/2"
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
</pre>
*Main '''if''' statement determines whether an Echo/AntiEcho or Real/Imaginary pair is recorded or skipped. The information is taken from the '''vdlist''' file which is generated by a separate script before running the experiments. If the current value of '''vd''' is 1u, the pair is skipped (goto 100 statement), if it is 3u, it is recorded. This addition replaces the text commented out and comes before the '''d1''' delay:<br>
<pre>;1 ze
; d11 pl16:f3
;2 d11 do:f3
;3 d12
;********Added for NUS*******
1 ze
d11 pl16:f3
2 d11 do:f3
short*2
3 short*5
4 1u
if "vd < 2u" goto 100
short*6
5 short
6 d11
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
d1
</pre>
*The final modification comes at the end of the pulse program and uses explicit looping (old style Bruker pulse programming). Note that F1PH and F2EA statements are commented out and the phase and delay increments or decrements are stated explicitly. These increments or decrements need to be executed even if the particular fids are skipped
<pre> go=2 ph31 cpd3:f3
;********Added for NUS*******
d11 do:f3 wr #0 if #0 zd
;----------------------------
100 short igrad EA  ; even if the pair is skipped, phases have to be increment appropriately.
short ip5*2  ; especially important if Complex or States-TPPI mode used for this dimension
lo to 3 times 2  ; end of Echo-AntiEcho loop

short id10  ; increment delay and phases for 15N
short ip3*2
short ip6*2
short ip31*2
short ivd  ; check the next value of vd
lo to 4 times l2  ; go back to beginng with the new vd value

short rd10  ; reset 15N delays and phases
short rp3
short rp6
short rp31
short ip8  ; increment 1H-indirect phases
short ip9
lo to 5 times 2

short id0  ; increment 1H-indirect delay.
lo to 6 times l3
1m
;^^^^^^^^^^^^^^^^^^^^^^^^^^^^
; d11 do:f3 mc #0 to 2
; F1PH(rd10 & rp3 & rp6 & rp31 & ip8 & ip9, id0)
; F2EA(igrad EA & ip5*2, id10 & ip3*2 & ip6*2 & ip31*2)
exit
</pre>
A good number of pulse programs are already modified and are available upon request from the Arrowsmith group (agutmana@uhnres.utoronto.ca):
{| cellspacing="1"
|
* HNCO (hncogp3d.nus)
* HNCA (hncagp3d.nus)
* CBCA(CO)NH (cbcaconhgp3d.nus)
* HBHA(CO)NH (hbhaconhgp3d.nus)
|
* <sup>15</sup>N-NOESY-HSQC (noesyhsqcfpf3gpsi3d.nus)
* <sup>13</sup>C-NOESY-HSQC (noesyhsqcetgp3d.nus)
* H(C)CH-TOCSY (hcchdigp3d3.nus)
* (H)CCH-TOCSY (hcchdigp3d2.nus)
|}

__NORICHEDITOR__

=== Generating the sampling schedule and running the experiment ===
__NORICHEDITOR__

Once the experiment is otherwise set up and the pulse program modifications are in place, the last step before running the experiment is to generate the sampling schedule. Depending on whether your computer runs Linux or Windows, the generator differs slightly (mainly in the usage of forward and backslashes). Both scripts are available upon request from the Arrowsmith group (together with the above pulse programs). The corresponding script should be renamed '''sparse.py''' and put in '''/opt/topspin/exp/stan/nmr/py/user/''' (Linux) or '''C:\Bruker\TOPSPIN\exp\stan\nmr\py\user\''' (Windows) on your computer.<br>

It can be called by typing '''sparse''' from the command prompt.

[[Image:TopspinNUSSnap0.png|thumb|center|1050px|Figure 1. Changing the pulse program and calling sparse.py script]]

After the initial greeting, you will be guided through a few steps as shown below.

*All vdlist files should be copied to a standard directory. If the suggested directory is wrong, please modify.

[[Image:TopspinNUSSnap2.png|frame|center|400px|Figure 2. Where the new vdlist should be placed.]]

*Main relevant parameters are presented in this window. The level of "sparsing" allows to fine tune the acquisition time. It should generally be 30% or more. T2sp parameters dictate the exponential biasing of the sampling and only need to be approximate. In the Arrowsmith group we use 20ms for 13C, 35 ms for 1H and 50ms for 15N. Usually the values are guessed correctly, but you can modify them if desired.

[[Image:TopspinNUSSnap3.png|frame|center|620px|Figure 3. Providing the parameters. Level of "sparsing" will give the number of FIDs actually recorded.]]

*Concluding window shows the final information on the number of FIDs to be recorded. Note that the experimental time shown by expt command will be wrong and should be multiplied by the "level of sparsing" parameter to give a correct estimate.

[[Image:TopspinNUSSnap4.png|frame|center|Figure 4. Checking that the file is copied to the right place and that the number of FIDs makes sense.]]

*To run the experiment type '''zg''' or '''multizg''' as the case may be. To process the spectrum you will need the '''vdlist''' file in addition to the usual parameter files.

=== Special Remarks ===

The script and the sampling schedule have a few features, worthy of some remarks:

*The first increments in each indirect dimension are sampled completely. This assists later in processing the data.
*The first plane in the "fast" dimension (usually 23 or XY) can be processed with TOPSPIN as if you were running a regular spectrum (i.e. with xfb).
*The first plane of the "slow" dimension (usually 13 or XZ) however cannot be processed in TOPSPIN unless the raw data is unscrambled first.
*Command expt gives incorrect experimental time estimate (see the final step above).
*An interrupted experiment cannot be continued by typing "go".
*The generated vdlist is named by default as follows "v"+date+"_"+ExpNum. Usually this should not cause problems, unless you wish to run two NUS experiments on the same day in the same experiment folder (number).
*The vdlist should have TD<sub>Y</sub>*TD<sub>Z</sub>/2 entries as each entry corresponds to recording or skipping two FIDs (Re/Im or Echo/AntiEcho). Bruker convention to record all increments in the fast dimension and only then increment the slow means that the selection for each slow plane appears twice (once for Real and once for Imaginary point to ensure proper quadrature detection in the slow indirect dimension). This is the reason you should rerun the script if you change these two parameters, as otherwise the data will be difficult to unscramble.<br>
*The 2 T2sp parameters give the exponential bias of the sampling in the two indirect dimensions. We did not investigate whether this value has an effect on dimensions with constant time evolution.

The PINE Server

2010-01-06T00:15:59Z

Agutmana: moved The PINE Server to Resonance Assignment/The PINE Server

#REDIRECT [[Resonance Assignment/The PINE Server]]

Resonance Assignment/The PINE Server

2010-01-06T00:15:59Z

Agutmana: moved The PINE Server to Resonance Assignment/The PINE Server

== '''Introduction''' ==

The PINE program (Probablistic Interaction Network of Evidence), recently developed by Arash Bahrami (NMRFAM, U. of Wisconsin), uses a novel aglorithm for the automated assignment of both backbone and side chain protein NMR resonances (Ref. 1).  PINE is available from a freely accessible [http://miranda.nmrfam.wisc.edu/PINE/ web server]. It is reported to have > 90% accuracy for backbone resonances and > 80% for aliphatic side chain resonances.

== '''Using the PINE Server''' ==

=== '''Input Files''' ===

*amino acid sequence of the protein target either in single-letter or three-letter format.
*spectral peak lists.  The program can accept peak lists in several formats including:
*SPARKY
*XEASY
*PINE
*additional information including:
*pre-assigned resonances
*amino acid selective labelling information
*grouped spins systems

<br>

=== The PINE Server ===

The PINE server is relatively simple to use (Figure 1).  The user uploads the protein sequence, spectral peak lists and any additional information and simply submits to the server.  There are extensive help links for the user on this main page.  Note that for Sparky peak lists, the current version of the server (1.0) cannot tolerate the Notes column; so, remove this column prior to submission.  The server returns the results by e-mail normally within a few minutes.  If you have a problem, [mailto:mani@nmrfam.wisc.edu e-mail] the Mani team.<br>

==== Figure 1:  The PINE Server ====

[[Image:PINEserver.png]]<br>

=== '''PINE Output''' ===

The user is e-mailed a list of PINE results files, as well as a zip file for download containing the entire output (Figure 2).

The PINE output files include:

*pecan_figure.jpg:  shows the predicted secondary structure elements across the sequence
*protein.jpg:  a small figure showing the completeness of the assignment across the sequence
*protein.pis:  a text file showing the assignments and percent confidences over the entire sequence.  This is particularly useful to study for any missing assignments.
*the assignments in BMRB format
*assigned peak lists in Sparky format

==== Figure 2:  PINE Results ====

[[Image:PINEresults.png]]

== '''References''' ==

1.    Bahrami, A., Assadi, A.H., Markley, J.L.  and Eghbalnia, H.R. (2009) Probabilistic interaction network of evidence algorithm and its application to complete labeling of peak lists from protein NMR spectroscopy. ''PLoS Comput. Biol. 5'', e1000307.

Resonance Assignment/Abacus/Spin systems identification

2010-01-06T00:00:10Z

Agutmana: /* Figure 4.1B,C */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==

1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] can be identified using the following spectra
<div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div><div>'''<span> Figure 4.1A</span>'''<span>[[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] } </span>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|500px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|500px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:59:56Z

Agutmana:

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==

1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] can be identified using the following spectra
<div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div><div>'''<span> Figure 4.1A</span>'''<span>[[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] } </span>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|5000px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|500px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:59:25Z

Agutmana:

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==

1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] can be identified using the following spectra
<div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div><div>'''<span> Figure 4.1A</span>'''<span>[[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] } </span>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:58:40Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==

1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] can be identified using the following spectra
<div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div><div>'''<span> Figure 4.1A</span>'''<span>[[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div><span /></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] } </span>

<br>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:57:22Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:55:43Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
<div><span></span></div><div><span> 1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] <span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
Figure 4.1A
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:55:21Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
</span></div><div><span> 1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] <span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
Figure 4.1A
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]  </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:54:29Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] <span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
Figure 4.1A</span>
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]     </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:53:46Z

Agutmana: /* Step 1. Generate HN-rooted spin-systems (bPB-fragments) */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
=== 1.1[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]] === <span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
Figure 4.1A</span>
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]     </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:52:52Z

Agutmana: /* Figure 4.1A */

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
<div> </div><div><span>          1.1  </span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
Figure 4.1A</span>
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]     </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:51:10Z

Agutmana:

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
<div> </div><div><span>          1.1  </span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
=== <span>Figure 4.1A</span> ===
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]     </span></div><div><span>
</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Water refinement setup and analysis

2010-01-05T23:49:20Z

Agutmana: moved Water refinement setup and analysis to Resonance Assignment/Abacus/Water refinement setup and analysis

#REDIRECT [[Resonance Assignment/Abacus/Water refinement setup and analysis]]

Resonance Assignment/Abacus/Water refinement setup and analysis

2010-01-05T23:49:20Z

Agutmana: moved Water refinement setup and analysis to Resonance Assignment/Abacus/Water refinement setup and analysis

*open new project PRJ4 { [[FMCGUI commands#Project.3ENew|Project>new]] } or
<div>     load old one PRJ3 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*set up water refinement calculations of the ensemble of structures obtained by CYANA  { [[FMCGUI commands#Structure.3EWater_refinement.3Ecalculate|Structure>Water Refinement>calculate]] }. The popped up window allows user to select file with cyana structural ensemble (final.pdb), cyana dihedral angle constraints (belok.aco), cyana distance constraints (final.upl) , H-bond constraints (hbond.upl), RDC data,  [[FMCGUI commands#Structure.3EAdd_ZN_ligands|ZINC ligands file]], and to specify cis-Proline residues and proton state of HIS residues. The command will set up the water refinement calculations in the user specified directory WRdir.
*curry out water refinement calculations (on linux cluster is recommended) following the instruction given in the project main window
*analyze and superimpose refined structures { [[FMCGUI commands#Structure.3EWater_refinement.3Esummary|Structure>Water Refinement>Summary]] }. The refined structural models are superimposed and combined in one file. Also, for each refined structure, different energy component are calculated and analysis of constraint violations is performed. The results of this analysis are placed in the created directory WRdir_results.
</div>

Structure calculation setup and analysis

2010-01-05T23:49:04Z

Agutmana: moved Structure calculation setup and analysis to Resonance Assignment/Abacus/Structure calculation setup and analysis

#REDIRECT [[Resonance Assignment/Abacus/Structure calculation setup and analysis]]

Resonance Assignment/Abacus/Structure calculation setup and analysis

2010-01-05T23:49:04Z

Agutmana: moved Structure calculation setup and analysis to Resonance Assignment/Abacus/Structure calculation setup and analysis

=== Step 1. Load Data ===
<div> </div>
**open new project PRJ3 { [[FMCGUI_commands#Project.3ENew|Project>new]] }
**load 15N NOESY peak list { [[FMCGUI commands#Data.3EN15_NOESY.3E|DATA>N15 NOESY>load]] }
**load 13C_aliphatic NOESY peak list  { [[FMCGUI commands#Data.3EC13_NOESY.3E|DATA>C13NOESY H2O>load]] }
**load 13C_aromatic NOESY peak list  { [[FMCGUI commands#Data.3EArom_NOESY.3E|DATA>AromNOESY>load]] }
**set tolerances  { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] } <span />
**<span>load ''<u>assigned </u>''chemical shifts  { [[FMCGUI commands#Fragment.3ELoad.3Eassigned|Fragment>Load>assigned]] }
</span>
<div><span>            The file with assigned chemical shifts could be either in “standard” format   ([[Spin systems format|assigned AA-fragments]]) or in [[Spin systems format|cyana format]] (prot-file).</span></div><div> </div><div><span>      </span></div>

=== Step 2. Set up constraints ===
<div> </div><div>''' '''The structure calculation requires dihedral angle constraints in the cyana format (aco-file). These constraints are usually prepared using the results of dihedral angle prediction by TALOS. H-bond constraints are optional.</div><div> </div>
*<span><span>        </span></span>calculate dihedral angle constraints { [[FMCGUI_commands#Structure.3EConstraints.3ETalos.3Ecalculate|Structure>Constraints>Talos>Calculate]] } 
*<span><span>        </span></span>set up H-bond constraints { [[FMCGUI_commands#Structure.3EConstraints.3EH-bonds.3ESpecify|Structure>Constraints>H-bonds>Specify]] } 
<div><span>            </span></div><div>In the cased dihedral angle or H-bond constraints in cyana format (aco-file or upl-file, respectively) already are  prepared,  then the constraints can be loaded  from the corresponding files <span>   { [[FMCGUI_commands#Structure.3EConstraints.3ETalos.3Eload|Structure->Constraints->Talos>Load]] } or { [[FMCGUI_commands#Structure.3EConstraints.3EH-bonds.3ESpecify|Structure>Constraints>H-bonds>Load]] } </span></div><div> </div><div> </div>

=== Step 3. Specify ligands coordinating ZN ions (if there are any) ===
<div> </div><div>If there are ZN ions as a part of a protein structure the file “zn_ligands” should be present inside FMCGUI project directory. This file can be created by the following command</div><div>''' '''</div>
*<span>    specify residues that coordinate ZN ion(s) { [[FMCGUI commands#Structure.3EAdd_ZN_ligands|Structure>Add ZN ion]] }
</span>
<div>''' '''</div><div> </div>

=== '''Step 4. Set up CYANA calculations''' ===
<div>''' '''</div>
*<span>    setting up structure calculations with CYANA  { [[FMCGUI commands#Structure.3ECalculate.3ECyana|Structure>Calcuate>cyana]] }
</span>
<div>'''<span>          </span>'''All files that are necessary for CYANA run are prepared and saved in the user specified directory, crun#, which is located inside the project directory. These files include chemical shifts (belok.prot file), sequence file, peak lists, dihedral angles constraints (file belok.aco), H-bond constraints, if available, (file hbond.upl), and constraints for ZN ions, if present, (files zn.upl, zn.lol).</div><div> </div>

=== Step 5. Structure evaluation and peak lists refinement ===
<div> </div><div>RPF analysis and DP score allow one to estimate goodness-of-fit of a structural ensemble to NOESY peak lists. The results of RPF analysis can serve both for structure validation and peak lists refinement. </div><div> </div>
*<span><span>      </span></span>run RPF analysis { [[FMCGUI commands#Structure.3ERPF.3ERP|Structure>RPF>RP]] }. The results of the RPF analysis include peak lists in the SPARKY format of both false negative and false positive peaks for C13-aliphatic NOESY, C13-Aromatic NOESY, and N15-NOESY spectra in separate files.
*<span><span>     </span></span>set up DP-score calculations with AutoStructure  { [[FMCGUI commands#Structure.3ERPF.3EDP|Structure>RPF>DP]] }.

Sequence specific assignment of PB fragments

2010-01-05T23:48:39Z

Agutmana: moved Sequence specific assignment of PB fragments to Resonance Assignment/Abacus/Sequence specific assignment of PB fragments

#REDIRECT [[Resonance Assignment/Abacus/Sequence specific assignment of PB fragments]]

Resonance Assignment/Abacus/Sequence specific assignment of PB fragments

2010-01-05T23:48:39Z

Agutmana: moved Sequence specific assignment of PB fragments to Resonance Assignment/Abacus/Sequence specific assignment of PB fragments

=== <span> Step 1. Peak picking of NOESY spectra</span> ===
<div><br></div><div><span>         To facilitate peak-picking of NOESY spectra, you can first generate expected tocsy peaks of PB-fragments using FMCGUI:   </span></div>
*<span><span>     </span></span>open project PRJ2 { [[FMCGUI commands#Project.3ELoad|Project>Load]] }
*<span>     generate tocsy peaks of N15 NOESY spectrum { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>N15NOESY]] }</span>
*<span><span>     </span></span>generate tocsy peaks of C13NOESY spectrum {[[FMCGUI commands#Fragment.3EExpected_Peaks.3E|<span> Fragment>Expected Peaks>C13NOESY</span>]] }
<div><span>       Then, using SPARKY, read the expected peaks into corresponding spectra and complete peak picking manually.</span></div><div> </div>

=== <span>Step 2. Probabilistic assignment of PB fragments to protein sequence</span> ===
<div></div><div>According to [[Introduction to ABACUS#Figure_1.3|FMC procedure]] you have to do the following:</div>
#<span>    Probabilistic typing of PB fragments</span>
#<span>    </span>Calculate  two fragments [[FMCGUI objects#Contact_map|contact maps]] C<sub>NOE </sub>and C<sub>HNCA . </sub>C<sub>NOE</sub> is a contact map based on 15N_ and 13C_NOESY data (it could be calculated by 2 different methods, “abacus” and “fawn”, respectively) and C<sub>HNCA</sub> is a map based on HNCA data.
#<span>   </span>Calculate [[FMCGUI objects#Assignment_probabilities|assignment probabilities]] <span>by Simulated Annealing (SA) or Replica Exchange Method (REM) Monte Carlo simulations</span>
<div> </div><div><span>     This can be done by the following commands:</span><span>     </span></div>
*   open project PRJ2 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*   load 15N NOESY peak list { [[FMCGUI commands#Data.3EN15_NOESY.3E|DATA>N15 NOESY>load]] }
*   load 13C NOESY peak list { [[FMCGUI commands#Data.3EC13_NOESY.3E|DATA>C13NOESY H2O>load]] }
*   set tolerances  { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }
*   calculate typing probabilities for all fragments { [[FMCGUI commands#Fragment.3EType.3ECalculate.3E|Fragment>Type>calculate>abacus]] }
<div><span>        You have to consider the warning messages shown in the project [[FMCGUI objects#Main_window|main window]] and to analyze/modify typing probabilities manually by using “Fragment Property Modification” (FPM) window. To open FPM window use command { [[FMCGUI commands#Fragment.3EType.3Efix|Fragment>Type>fix]] }. This window has 3 sections. Top section allows you to select a fragment (by user ID) and modify its typing probabilities. The middle section shows typing probabilities that correspond to the selected amino acid type for all fragments. Here you can fix for any fragment it’s  typing probability corresponding to the selected amino acid type to the values of 1 or 0. </span></div><div> </div>
*  calculate fragment contact map from HNCA data { [[FMCGUI commands#Assignment.3EContacts.3EHNCA|Assignment>Contacts>HNCA]] }         

      Contact map C<sub>HNCA</sub> is calculated and loaded in the memory. It is strongly recommended to check the messages in the project main window regarding HNCA peak list. In case there are inconsistencies present in the input HNCA peak list, go back to spectra and fix the list. 

*<span>  calculate fragment contact map from NOESY data { [[FMCGUI commands#Assignment.3EContacts.3ENOE.3Eabacus|Assignment>Contacts>NOE>abacus]] }. In the result, two contact maps, </span>C<sub>NOE_B</sub> and C<sub>NOE_F </sub>are calculated and loaded in the memory. Calculation of <span> </span>C<sub>NOE_B</sub>  makes use of BACUS procedure for NOESY data interpretation, while calculation of <span> </span>C<sub>NOE_F</sub> <sub> </sub> does not.<span><span>  </span></span>
*<span><span /></span>Calculate assignment probabilities.    It can be done using two different Monte Carlo simulation methods. Namely, Simulating Annealing (SA) { [[FMCGUI commands#Assignment.3ECalculate_Probabilities.3ESA|Assignment>Calculate Probabilities>SA]] } and Replica Exchange Method (REM) { [[FMCGUI commands#Assignment.3ECalculate_Probabilities.3ESA|Assignment>Calculate Probability>REM]] }. Before starting calculations you have to specify control parameters. The main parameters to consider are: ‘''Name of the SA/REM run''’, ‘''Size of the pool for unassigned fragments''’, ‘''number of SA runs''’, ‘''Final Temperature''’ (SA), ‘''Low Temperature''’ (REM), and ‘''NOE contact map''’. The results of the calculations will be stored in the directory under specified name which is created inside PRJ2/assign/ directory. The main result consists of optimal and sub-optimal fragments assignments and assignment probability map. The last one will be automatically loaded in the memory as well.
<div><span>         Calculation of assignment probabilities could be repeated a few times using different methods and parameters. Result of each calculation is stored in a separate directory. Therefore, there could be a few different directories (for example, sa_run1, sa_run2, rem_run0, rem_run1, rem_run3… ) within PRJ2/assign/ directory that contain different assignment probability maps.</span></div><div> </div>

=== Step 3. Sequence-specific assignment of PB fragments by analyzing probabilities ===
<div> </div><div><span>   A fragment assignment to a sequence position using FMCGUI could be done in two ways, manually and using assignment probability map, respectively. </span></div><div> </div>
*<span>   manual assignment { [[FMCGUI commands#Assignment.3EFix_Assignment.3EManually|Assignment>Fix Assignment>manually]] }. </span>
<div>This command pops up ‘Fragment Property Modification’ window. You can change the assignment ID of any selected fragment using the bottom section of the window. </div><div> </div>
*<span>  assignment using probability map  { [[FMCGUI commands#Assignment.3EFix_Assignment.3EUsing_Probability_map|Assignment>Fix Assignment>using probability map]] }. You have to select a calculation directory (sa_run# or rem_run#) that contains [[FMCGUI objects#Assignment_probabilities|assignment probability]] map, P<sub>SA</sub> or P<sub>REM</sub> </span><span>, </span>and to specify the probability threshold  P<sub>min</sub>. A fragments ''f'' will be assigned to position ''s'' if the condition <span>P<sup>s</sup>(f)<P<sub>min</sub>   is satisfied (see [[Introduction to ABACUS#Figure_1.4|Figure 1.4]]).</span>
<div> </div>

=== Step 4. Assignment analysis ===
<div> </div><div>In the case of poor data, only a part of the fragments get assigned unambiguously. The uncertainty in fragments assignment could be resolved manually with the help of FMCGUI command { [[FMCGUI_commands#View.3EAssignment|View>Assignment]] }. This command pops up “Assignment Graph” window that provides you with graphical tool to visualize the current assignment and to analyze sub-optimal fragment assignments.</div><div> </div>

=== Step 5. Final resonance assignment ===
<div> </div><div>When sequence specific PB fragment assignment is done you have to put in order assigned fragments and to assign CO resonances using command { [[FMCGUI commands#Fragment.3EModify_assigned|Fragment>Modify assigned]] }.</div><div><span>           </span></div><div><br></div>

Spin systems identification

2010-01-05T23:48:23Z

Agutmana: moved Spin systems identification to Resonance Assignment/Abacus/Spin systems identification

#REDIRECT [[Resonance Assignment/Abacus/Spin systems identification]]

Resonance Assignment/Abacus/Spin systems identification

2010-01-05T23:48:23Z

Agutmana: moved Spin systems identification to Resonance Assignment/Abacus/Spin systems identification

<div>Normally, the NMR spectra shown in [[Introduction to ABACUS#NMR_spectra_required_for_ABACUS|Table 1]] are collected for ABCUS. Spectra should ideally be collected from the same protein preparation and under the same conditions to make sure peaks are within tolerance between spectra. All spectra need to be appropriately synchronized <span>and calibrated against reference spectra of your choice. It is a good idea to use as a reference for spectra calibration the 2D HN-projection of 15N-NOESY and the 2D CH-projection of the 13C-NOESY. </span></div><div> </div>
== Step 1. Generate HN-rooted spin-systems (''b''PB-fragments) ==
<div> </div><div><span>          1.1  </span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> can be identified using the following spectra </span></div><div><br><span>             15N_HSQC</span></div><div><span>             HNCO</span></div><div><span>             CBCA(CO)NH</span></div><div><span>             HBHAC (CO)NH</span></div><div><span>             HNCA</span></div><div><span>            15N_NOESY</span></div><div> It is a good idea to analyze all these spectra simultaneously, for example,  using SPARKY, in order to obtain  peak lists of 15N_HSQC, HNCA,  CBCA(CO)NH, and HBHA(CO)NH spectra (see Figure 4.1A). The first two spectra should be referenced.</div><div><span> </span></div>
=== <span>Figure 4.1A</span> ===
<div><span>   [[Image:Fmcgui Fig4.1a.jpg|thumb|center|600px]]     </span></div><div><span>

</span></div><div><span>        1.2. Create initial HN-rooted spin-systems (</span>[[Introduction to ABACUS#bPB_fragment|''b''PB-fragments]]<span> </span><span>) using FMCGUI:</span></div><div> </div>
*<span><span>       </span></span>start new project PRJ_1 { [[FMCGUI commands#Project.3ENew|Poject>new]] }
*<span><span>       </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>       </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }<br>
*<span><span>       </span></span>load referenced CBCA(CO)NH peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACONH>load]] }<br>
*<span><span>       </span></span>load referenced HBHA(CO)NH peak list { [[FMCGUI commands#Data.3EHBHACONH.3E|DATA>HBHACONH>load]] }
*<span><span>       </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }<br>
*<span><span>       </span></span>set tolerances for matching of resonances in different spectral dimensions { [[FMCGUI commands#Data.3ETolerances|Data>Tolerances]] }<br>
*<span><span>       </span></span>create bPB-fragments { [[FMCGUI commands#Fragment.3ECreate.3Efawn|Fragment>create>fawn]] }<br>
<div>First, a referenced C13_hsqc peak list is created and shown in new window ‘fake C13 HSQC’. Warning messages are shown in the project main window. You have to check the list and modify it if needed. Then, press ‘OK’ button in the ‘‘fake C13 HSQC’ window. In the result, new window ‘Create Fragment’ pops up. The window consists of three sections. The left sections contains suggested ''b''PB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Consider warning messages shown in the project main window and check/modify generated ''b''PB-fragments in the left section of “Create Fragment” window. When satisfied, the ''b''PB-fragments will be loaded in the memory by clicking on “OK” button.</div>
*<span>       save project PRJ_1. { [[FMCGUI commands#Project.3ESave|Project>Save]] } or  { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>

<br>

<br>

<br>

<br>
<div><span>     </span></div>

== Step 2. Complete PB-fragments ==

    At this step ''b''PB-fragments are completed with aliphatic side-chain resonances and  additional spin-systems (without HN) are identified using the following spectra.
<div> </div><div><span>               13C-HSQC</span></div><div>''<span>               </span>''(H)CCH-TOCSY</div><div><span>               H(C)CH-TOCSY</span> </div><div><span>               13C-NOESY</span></div><div> </div><div>        2.1. Generate expected peak lists for C13HSQC, (H)CCH-TOCSY, and H(C)CH-TOCSY spectra using created ''b''PB fragments:</div><div> </div>
*    open project PRJ1 { [[FMCGUI commands#Project.3ELoad|Project>load]] }
*    generate expected C13hsqc_exp.list that consists of C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub> moieties<span>   { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>C13HSQC]] }

</span>

*    generate expected CCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>(H)CCH]] }<br>
*    generate expected HCH-Tocsy.list { [[FMCGUI commands#Fragment.3EExpected_Peaks.3E|Fragment>Expected Peaks>H(C)CH]] }<br>
<div> </div><div><span>        2.2<span>    </span></span>Read the generated peaks into SPARKY. </div><div> </div><div><span>        2.3<span>    </span></span>Using SPARKY, complete ''b''PB fragments with aliphatic side-chain resonances by analyzing C<sub>a</sub>-H<sub>a</sub> and C<sub>b</sub>-H<sub>b</sub><span> strips in all CH_rooted spectra (see Figure 4.1B,C). New resonances should be peaked and referenced ''<u>only</u>'' in 13C_HSQC spectrum.</span></div><div> </div>
=== Figure 4.1B,C ===
<div></div><div></div><div>[[Image:Fmcgui Fig4.1b.jpg|thumb|left|440px]][[Image:Fmcgui Fig4.1c.jpg|thumb|right|440px]]</div><div></div><div></div><div></div><div><span>        </span></div><div><br></div><div><span>2.4<span>    </span></span>When all peaks corresponding to HN-rooted spin-systems are peaked in 13C_HSQC spectrum, the unpicked peaks are used as a starting point for identification of spin-systems without backbone HN resonances (PB fragments corresponding to residues before prolines in the protein sequence, last residue and residues missing in HN-rooted spectra).</div><div> </div>
<br>

== Step 3. Spin-systems validation and correction ==
<div> </div><div>Validate PB-fragments using FMCGUI:</div>
*<span>        start a new project PRJ2 { [[FMCGUI commands#Project.3ENew|Project>new]] }
</span>

<br>

*<span><span>        </span></span>load protein sequence { [[FMCGUI commands#Data.3EProtein_Sequence.3ELoad|DATA>Protein Sequence>load]] }
*<span><span>        </span></span>load referenced 15N_HSQC peak list { [[FMCGUI commands#Data.3EN15_HSQC.3E|DATA>N15 HSQC>load]] }
*<span><span>        </span></span>load referenced 13C_HSQC peak list { [[FMCGUI commands#Data.3EC13_HSQC.3E|DATA>C13 HSQC>load]] }
*<span><span>        </span></span>load referenced HNCA peak list { [[FMCGUI commands#Data.3EHNCA.3E|DATA>HNCA>load]] }
*<span><span>        </span></span>load  CBCA(CO)HN peak list { [[FMCGUI commands#Data.3ECBCACONHN.3E|DATA>CBCACOHN>load]] }
*<span><span>        </span></span>load  HNCO peak list { [[FMCGUI commands#Data.3EHNCO.3E|DATA>HNCO>load]] }
*<span><span>        </span></span>set tolerances for matching of resonances in different spectral dimensions  { [[FMCGUI commands#Data.3ETolerances|DATA>Tolerances]] }
*<span><span>        </span></span>create and validate PB fragments { [[FMCGUI commands#Fragment.3ECreate.3Eabacus|Fragment>create>abacus]] }
<div>This command starts script ‘''sps_create’''. In the result, a new window [[FMCGUI commands#Fragment.3ECreate.3Eabacus|"Create Fragment"]] pops up. The warning messages of the ‘''sps_create’'' script are shown in the main window and indicate spin-system that have low score (see [[Introduction to ABACUS#Figure_1.2|Figure 1.2]]), namely, spin=systems with S<sub>max</sub> < 10<sup>-4</sup>. Following the warnings check and modify, if necessary, generated PB fragments in the left section of “Create Fragment’ window. Alternatively, go back to spectra, fix peak lists accordingly, and repeat the fragment generation/validation again.</div><div>When satisfied, the PB fragments will be saved (file "sps_pb.dat" into directory "PRJ2/sps") and loaded in memory by pressing "OK" button in “Create Fragment” window.</div>
*<span>       save project PRJ2 { [[FMCGUI commands#Project.3ESave|Project>Save]] } or { [[FMCGUI commands#Project.3EQuit|Project>Quit]] }
</span>
<div>''' '''</div>
<br>

Resonance Assignment/Abacus

2010-01-05T23:48:05Z

Agutmana:

#[[Introduction to ABACUS]]
#[[FMCGUI objects]]
#[[FMCGUI commands]]
#FMCGUI Data Formats
#*[[Protein Sequence format]]
#*[[Peak Lists format]]
#*[[Spin systems format]]
#FMCGUI HOW-TOs
#*[[Spin systems identification]]
#*[[Sequence specific assignment of PB fragments]]
#*[[Structure calculation setup and analysis]]
#*[[Water refinement setup and analysis]]

Spin systems format

2010-01-05T23:35:33Z

Agutmana: moved Spin systems format to Resonance Assignment/Abacus/Spin systems format

#REDIRECT [[Resonance Assignment/Abacus/Spin systems format]]

Resonance Assignment/Abacus/Spin systems format

2010-01-05T23:35:33Z

Agutmana: moved Spin systems format to Resonance Assignment/Abacus/Spin systems format

<div>Not assigned spin-systems ([[Introduction to ABACUS#PB_fragment|PB fragments]]) could be loaded in FMCGUI using only one, "standad", format.</div><div>Assigned chemical shifts (spin-systems) could be loaded using two different formats, standard and cyana format, respectively.</div><div> </div>
__TOC__

=== '''1. PB-fragments in standard format.''' ===
<div></div><div></div><div></div><div>Each fragment is represented by a number of lines. The groups of lines corresponding to different fragments are separated by empty line. </div><div>The line that follow the last fragment should have 'Q' at the first position.</div><div> </div><div>A fragment is described as follows:</div><div></div><pre>31 X    7     0.000 
   9.025  HN    106.560  N
   5.221  HA     55.302  CA
   1.940  HB*    33.679  CB
   1.480  HG1    27.976  CG
   1.775  HG2    27.976  CG
   2.943  HD1    43.958  CD
   3.205  HD2    43.958  CD
</pre><div></div><div></div><div></div><div><span><font size="2"></font></span></div><div></div><div></div><div>    </div><div><span><font size="2"></font></span>Here, the first line specify user ID, 1-letter residue type ('X' if not known), the number of the following lines (regading the fragment), and chemical shift of backbone C' atom ( should be '0.000' if it is not known). </div><div> </div><div> The fragments user ID could be any digit number < 500. There is no restriction on the order of fragments in the input file.</div><div> </div><div>''<font size="2">Example</font>''<font size="2">1.1. Not assigned PB-fragments.</font></div><div><font size="2"></font></div><pre> 90 X    5     0.000

   8.462  HN    122.771  N
   4.262  HA     56.242  CA
   1.880  HB1    30.528  CB
   2.006  HB2    30.528  CB
   2.222  HG*    36.201  CG
   
 91 S    4   174.082
   8.089  HN    123.488  N
   4.626  HA     55.058  CA
   3.413  HB1    65.338  CB
   3.801  HB2    65.338  CB
   
247 X    5     0.000
   8.070  HN    123.607  N
   4.034  HA     62.358  CA
   2.048  HB     32.775  CB
   0.873  HG1*   21.111  CG1
   0.884  HG2*   20.501  CG2
   
 18 X    4     0.000
   8.110  HN    123.044  N
   5.042  HA     58.559  CA
   4.179  HB     70.135  CB
   0.087  HG2*   16.018  CG2
Q

</pre><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div>        </div><div>'''         '''</div>
=== '''2. Assigned AA-fragments in standard format.''' ===
<div>     </div><div></div><div></div><div></div><div></div><div></div><div>AA-fragments should be ordered in the input file according to their assignment to </div><div>protein sequence positions. </div><div>        </div><div></div><div>
Each fragment is described as follows
<pre> 11 T    4   174.109         106
   8.114  HN    113.206  N
   4.325  HA     61.900  CA
   4.283  HB     69.855  CB
   1.126  HG2*   21.639  CG2</pre>
 

 
</div><div></div><div></div><div></div><div>Here, the first line specifies  protein sequence position to which the fragment is assigned, 1-letter amino acid type, the number of the following lines describing the fragment, chemical shift of backbone C' atom, and user ID of the fragment.The last number, user ID, is optional.</div><div></div><div></div><div> </div><div>''<font size="2">Example</font>''<font size="2">2.1. Assigned AA-fragments with user ID specified.</font></div><div><font size="2">  </font></div><pre>...............................
 10 G    2   174.136         158
   8.605  HN    110.399  N
   3.708  HA*    46.225  CA
   
 11 T    4   174.109         106
   8.114  HN    113.206  N
   4.325  HA     61.900  CA
   4.283  HB     69.855  CB
   1.126  HG2*   21.639  CG2
   
 12 N    1   175.100         999
   8.231  HN    122.327  N
   
 16 A    2   179.269          50
   3.224  HA     56.082  CA
   1.518  HB*    18.860  CB
   
 17 D    3   179.378          74
   8.451  HN    116.577  N
   4.370  HA     57.335  CA
   2.656  HB*    39.698  CB
   
 18 V    5   177.276         117
   7.579  HN    122.993  N
   3.720  HA     65.701  CA
   2.068  HB     31.930  CB
   1.160  HG1*   22.719  CG1
   0.642  HG2*   20.806  CG2
............................  

</pre><div><font size="2">  </font></div><div><font size="2"> </font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font><div><font size="2">''<font size="2"></font>''</font></div></div>
<font size="2"><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div>''<font size="2"></font>''</div><div>''<font size="2">Example</font>''<font size="2">2.2. Assigned AA-fragments without user ID specified.</font></div><div><font size="2"></font></div><div><font size="2"> </font> </div><div><font size="2"></font></div><pre> .....
 10 G    2   174.136         
   8.605  HN    110.399  N
   3.708  HA*    46.225  CA
   
 11 T    4   174.109         
   8.114  HN    113.206  N
   4.325  HA     61.900  CA
   4.283  HB     69.855  CB
   1.126  HG2*   21.639  CG2
   
 12 N    1   175.100         
   8.231  HN    122.327  N
   
 16 A    2   179.269         
   3.224  HA     56.082  CA
   1.518  HB*    18.860  CB
   
 17 D    3   179.378         
   8.451  HN    116.577  N
   4.370  HA     57.335  CA
   2.656  HB*    39.698  CB
   
 18 V    5   177.276         
   7.579  HN    122.993  N
   3.720  HA     65.701  CA
   2.068  HB     31.930  CB
   1.160  HG1*   22.719  CG1
   0.642  HG2*   20.806  CG2
........... 

</pre><div><font size="2">     </font></div><div><font size="2">       </font></div><div><font size="2">     </font></div><div><font size="2"></font></div><div><font size="2"></font></div></font>

=== '''<span id="1259349916483S" style="display: none;"> </span>3. CYANA chemical shift file (prot-file).''' ===
<div>'''    '''</div><div></div><div></div><div>Assigned chemical shifts caould be loaded in FMCGUI and automatically transformed in a set of assigned PB-frsagments.</div><div>    </div><div></div><div></div><div></div><div></div><div></div><div>''<font size="2">Example</font>''<font size="2">3.1. Assigned chemical shifts.</font></div><div><font size="2">  </font></div><pre>    1   8.414   0.000 H       1
     2 123.795   0.000 N       1
     3   4.723   0.000 HA      1
     4  53.298   0.000 CA      1
     5   1.898   0.000 HB3     1
     6  32.288   0.000 CB      1
     7   2.030   0.000 HB2     1
     8   2.475   0.000 HG3     1
     9  32.024   0.000 CG      1
    10   2.547   0.000 HG2     1
    11 177.440   0.000 C       2
    12   4.379   0.000 HA      2
    13  63.455   0.000 CA      2
    14   1.914   0.000 HB3     2
    15  32.194   0.000 CB      2
    16   2.268   0.000 HB2     2
    17   1.971   0.000 HG3     2
    18  27.313   0.000 CG      2
    19   2.008   0.000 HG2     2
    20   3.650   0.000 QD      2
    21  50.670   0.000 CD      2
    22 173.945   0.000 C       3
    23   8.438   0.000 H       3
    24 108.915   0.000 N       3
    25   3.941   0.000 QA      3
    26  45.341   0.000 CA      3
.................................. </pre><div><font size="2">  </font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div><font size="2"></font></div><div></div><div></div>

Peak Lists format

2010-01-05T23:35:18Z

Agutmana: moved Peak Lists format to Resonance Assignment/Abacus/Peak Lists format

#REDIRECT [[Resonance Assignment/Abacus/Peak Lists format]]

Resonance Assignment/Abacus/Peak Lists format

2010-01-05T23:35:18Z

Agutmana: moved Peak Lists format to Resonance Assignment/Abacus/Peak Lists format

__TOC__

= '''1. SPARKY format'''. =

Both referenced and not referenced [[FMCGUI objects#Peak_list|peak lists]] can be provided in this format as input for FMCGUI.<br>Peaks referencing, if present, has format F#, were # is user defined PB-fragment ID. 

The volume/height, if present,  could be provided by digit number or by float number in E format (0.141E+10)<br>

<br>

''Example 1.1''. Referenced HNCA  list:
<pre>     User         w1         w2         w3 

         F1     55.269    106.560      9.027
         F1     43.558    106.560      9.026
         F2     51.232    114.375      9.101
         F2     59.634    114.375      9.096
         F3     57.686    118.215      9.514
         F3     59.071    118.215      9.519
         F4     55.762    118.966      9.306
         F4     59.071    118.966      9.306
         F5     52.547    119.119      9.250
         F5     59.071    119.119      9.249
         F6     59.071    120.109      9.230
         F6     55.128    120.109      9.232
         F7     55.598    122.600      9.599
         F9     56.184    122.430      9.316
         F9     57.616    122.430      9.319
        F11     58.719    122.464      9.158
        F11     55.292    122.464      9.157
....................................
</pre>
<br>''Example 1.2''. Not referenced N15-NOESY list.
<pre>     Assignment         w1         w2         w3   Data Height

 ?-?-?      3.592    106.850      8.261      1435751
            ?-?-?      4.244    106.850      8.259      1096259
            ?-?-?      1.625    108.915      8.438       544482
            ?-?-?      6.898    108.915      8.438       428124
            ?-?-?      2.017    119.793      7.851       858875
            ?-?-?      4.358    119.793      7.855      1260421
            ?-?-?      4.573    126.081      8.518       590579
            ?-?-?      3.891    126.081      8.522       651350
            ?-?-?      5.665    125.092      8.261       793816
            ?-?-?      3.567    125.092      8.264      1123055
            ?-?-?      3.300    125.092      8.262       814461
            ?-?-?      4.713    125.092      8.262      6211645
            ?-?-?      6.976    125.092      8.262      2048095
            ?-?-?      7.884    125.092      8.261       651915
...................................................
</pre>
<br>''Example 1.3.'' Not referenced HNCO list.
<pre>      w1  w2      w3 

  176.935    106.560   9.025
  175.679    114.375   9.107
  175.310    118.215   9.517
  174.027    118.966   9.307
  178.232    119.102   9.255
  175.174    120.109   9.227
  173.781    122.600   9.599
  175.720    122.430   9.312
  172.143    122.464   9.165
  173.699    121.269   9.058
  176.102    121.525   9.099
  173.645    123.727   9.143
  173.781    124.819   9.113
..........................</pre>
''Example 1.4''. Referenced N15-HSQC list.
<pre>      User         w1         w2 

        F34    123.334      8.843
        F99    122.498      7.685
       F144    117.711      7.610
       F163    121.227      8.178
        F50    116.577      8.451
         F1    106.560      9.025
       F100    122.361      7.821
       F101    121.372      7.868
       F102    122.361      8.006
       F103    122.378      8.048
       F105    122.703      8.123
       F106    122.327      8.231
       F107    121.440      8.358
       F109    120.570      8.577
        F11    122.464      9.164
       F111    120.535      8.306
       F112    121.218      8.200
       F113    120.843      8.157
       F114    121.338      8.105
...........................
</pre>
<br>''Example 1.5''. Nor referenced CBCACONH list.
<pre>..........
</pre>
<br>

<br>

= '''2. XEASY format.''' =

Only not referenced peak lists could be provided in this format as input for FMCGUI.

The first line of the file should start with the character '#'.

<br>

''Example 2.1. ''Not referenced Arom-NOESY list.
<pre># Number of dimensions 3
#FORMAT xeasy3D
#INAME 1 C
#INAME 2 H
#INAME 3 HC
#CYANAFORMAT ChH
     1 132.896   7.221   7.216 1 U   0.141E+10  0.000E+00 e 0     0     0     0
     2 132.896   6.799   7.219 1 U   0.177E+09  0.000E+00 e 0     0     0     0
     3 132.896   4.695   7.217 1 U   0.205E+08  0.000E+00 e 0     0     0     0
     4 132.896   3.271   7.220 1 U   0.274E+08  0.000E+00 e 0     0     0     0
     5 132.896   3.006   7.219 1 U   0.180E+08  0.000E+00 e 0     0     0     0
     6 132.896   1.848   7.228 1 U   0.108E+08  0.000E+00 e 0     0     0     0
     7 132.896   1.436   7.233 1 U   0.112E+08  0.000E+00 e 0     0     0     0
     8 118.540   6.796   6.796 1 U   0.452E+10  0.000E+00 e 0     0     0     0
     9 118.540   7.221   6.792 1 U   0.228E+09  0.000E+00 e 0     0     0     0
    10 118.540   7.054   6.794 1 U   0.167E+09  0.000E+00 e 0     0     0     0
    11 118.540   4.708   6.795 1 U   0.641E+08  0.000E+00 e 0     0     0     0
    12 118.540   2.032   6.794 1 U   0.203E+08  0.000E+00 e 0     0     0     0
    13 118.540   0.537   6.797 1 U   0.380E+08  0.000E+00 e 0     0     0     0
    14 131.288   6.911   6.900 1 U   0.261E+09  0.000E+00 e 0     0     0     0
    15 131.288   7.170   6.899 1 U   0.897E+08  0.000E+00 e 0     0     0     0
............................................................................
</pre>
<br>

= <br>'''3. "Standard" ABACUS format.''' =

Only not referenced peak lists could be provided in this format as input for FMCGUI.

<br>

''Example 3.1''. Not referenced N15-NOESY list.
<pre>1   106.850   4.244   8.259   1096259
2   108.915   1.625   8.438   544482
3   108.915   6.898   8.438   428124
4   119.793   2.017   7.851   858875
5   119.793   4.358   7.855   1260421
6   126.081   4.573   8.518   590579
7   126.081   3.891   8.522   651350
8   125.092   5.665   8.261   793816
9   125.092   3.567   8.264   1123055
......................................</pre>
<br>''Example 3.2''. Not referenced C13-NOESY list.
<pre>1   61.234   2.083   4.157   0.124E+08
2   61.234   7.471   4.152   0.751E+07
3   31.252   4.219   2.974   0.814E+07
4   31.252   4.222   3.029   0.985E+07
5   50.670   4.728   3.651   0.114E+09
6   17.853   2.445   0.865   0.506E+08
7   17.853   3.055   0.868   0.189E+08
8   17.853   3.837   0.868   0.285E+08
9   17.853   7.122   0.873   0.278E+08
10   17.853   6.847   0.868   0.185E+08
11   17.853   8.292   0.860   0.263E+08
12   17.853   8.393   0.860   0.198E+08
........................................
</pre>

Resonance Assignment/Abacus

2010-01-05T23:35:08Z

Agutmana:

1. [[Introduction to ABACUS]]

2. [[FMCGUI objects]]

3. [[FMCGUI commands]]

4. FMCGUI Data Formats

          [[Protein Sequence format]]

          [[Peak Lists format]]

          [[Spin systems format]]

5. [[FMCGUI HOW-TOs]]

            [[Spin systems identification]]

            [[Sequence specific assignment of PB fragments]]

            [[Structure calculation setup and analysis]]

            [[Water refinement setup and analysis]]

     

Resonance Assignment/Abacus

2010-01-05T23:34:52Z

Agutmana:

1. [[Introduction to ABACUS]]

2. [[FMCGUI objects]]

3. [[FMCGUI commands]]

4. [[FMCGUI Data Formats]]

          [[Protein Sequence format]]

          [[Peak Lists format]]

          [[Spin systems format]]

5. [[FMCGUI HOW-TOs]]

            [[Spin systems identification]]

            [[Sequence specific assignment of PB fragments]]

            [[Structure calculation setup and analysis]]

            [[Water refinement setup and analysis]]

     

Resonance Assignment/Abacus

2010-01-05T23:34:35Z

Agutmana:

Protein Sequence format

2010-01-05T23:34:17Z

Agutmana: moved Protein Sequence format to Resonance Assignment/Abacus/Protein Sequence format

#REDIRECT [[Resonance Assignment/Abacus/Protein Sequence format]]

Resonance Assignment/Abacus/Protein Sequence format

2010-01-05T23:34:17Z

Agutmana: moved Protein Sequence format to Resonance Assignment/Abacus/Protein Sequence format

__TOC__

== '''1. Fasta format.''' ==

The first line should start with '>' symbol.

Next one or more lines contain sequence in 1-letter code.

<br>

''Example 1.1:<br>''
<pre>>
MDSKEVLVHVKNLEKNKSNDAAVLEILHVLDKEFVPTE
KLLRETKVGVEVNKFKKSTNVEISKLVKKMISSWKD </pre>

== <br>'''2. "Standard" format.''' ==

Each line contains name of one residue in 3-letter code and, optionally, the residue ID.

(Only residue ID of the first residue is used to start [[FMCGUI objects#Protein_sequence|numbering of sequence positions]].

<br>

''Example 2.1.'' (position ID is not specified):
<pre>GLN
GLY
HIS
MET
PRO
GLY
ILE
ILE
TYR
GLU
GLY
.... </pre>
''Example 2.2.'' (with specified all position ID):
<pre>GLN -3
GLY -2
HIS -1
MET 0
PRO 1
GLY 2
ILE 3
ILE 4
TYR 5
..... </pre>
''Example 2.3.'' (with specified first position ID):
<pre>GLN -3
GLY
HIS
MET
PRO
GLY
ILE
.....</pre>

FMCGUI commands

2010-01-05T23:33:32Z

Agutmana: moved FMCGUI commands to Resonance Assignment/Abacus/FMCGUI commands

#REDIRECT [[Resonance Assignment/Abacus/FMCGUI commands]]