NESG Wiki - User contributions [en]

Resonance Assignment/The PINE Server

2010-04-27T15:34:00Z

Jlmills:

== '''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://pine.nmrfam.wisc.edu/ 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

 

=== 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. 

==== Figure 1:  The PINE Server ====

[[Image:PINEserver.png]] 

=== '''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.

Side chain assignment with CN-NOESY in XEASY

2009-12-18T17:52:13Z

Jlmills:

One can go to the simultaneous 15N,13C-resolved [1H, 1H]-NOESY directly rather than the HCCH-spectra to complete the side-chain assignment after backbone and HB resonance assignment were obtained. Separately recorded 15N, 13C(aliphatic), and 13C(aromatic) NOESYs can also be combined and used as a simultaneous NOESY, however, extra effort may be needed. Although the risk is low, it is suggested to confirm the assignment from NOESY by using HCCH due to the risk of misinterpretion of inter-residue NOE as intra-residue NOE.

*Pros
**Fast. Since it is necessary to double check the chemical shift in NOESY spectra before NOE peak picking and interpretion in NOESY, the side chain assignment can be done while check the chemical shift in NOESY and will not incease much extra time.
**Redundant information. In addtion to the correlations observed in HcCH-COSY/TOCSY type spectra, NOE of side-chain resonances to intra-and sequential-amide resonances provides addtional information for peak pattern recognization, which also reduce the risk.
*Cons
**Redundant information. There is risk of misinterpretation of inter-residue NOE as intra-residue.
**May need experience.

 One can start side-chain assignment as following after correct backbone assignment and HA/HB assignments were obtained.

#Process the simultaneous NOESY as a single 13C NOESY spectra. In this case, the 15N chemical shift is calibrated as 13C shift. The real chemical shift can be easily calculate back based on the acquisition parameters. It is recommended to analyze the simultaneous NOESY as a real single NOESY.
#In UBNMR, run <tt>makeNoePeaks</tt> to generate a starting simultaneous NOESY peakList including intra- and sequential NOEs considering previous assigned backbone and HA/HB chemical shifts and also averaged chemical shifts from BMRB for all assignable unassigned side-chain resonances. The 15N chemical shift has already been converted to pseudo-13C chemical shift which will exactly fit the spectra.
#In XEASY, use <tt>ns</tt> to load two copies of the Simultaneous 15N,13C-resolved (1H,1H) NOESY and display the two copies in orthogonal views (X-axis :w1(13C/15N), Y-axis: w2(1H); Z-axis: w3(1HC/HN) & X-axis: w3(1HC/HN), Y-axis: w2(1H); Z-axis: w1(13C/15N) use <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt>, respectively, to load noe.seq, bbsc.prot and simnoesyI1.peaks; use <tt>sp</tt>, <tt>se</tt> and <tt>gs</tt> to display [w2(1H),w3(1H)]-strips and [w2(1H),w1(13C)]-strips residue by residue.
#In XEASY, start from one view (X-axis: w1(13C/15N), Y-axis: w2(1H); Z-axis: (1HC/HN)) of the NOESY, check [w2(1H),w3(1HN)] (both intra- and sequential)-[w2(1H),w3(1HA)]-[w2(1H),w3(1HB)]-strips to confirm assignments for backbone and HB resonance. Adjust peak position and resort strips if necessary. Do same check in the other view (X-axis: w1(13C/15N), Y-axis:w2(1H); Z-axis: w3(1HC/HN)). '''Example Figure 1 for Ile 12 side-chain assignment by using NOESY''' This is how it looks after this step. The strips are displayed in the order of HN, N, HA, CA, HB, CB, QG2, CG2, HG12, CG1, HG13, CG1, QD1, CD1, HN (i+1) and N (i+1), where the name represent the x-dimesion of the strip, i+1 stands for the next residue. Since the assignment of HN, N, HA, CA, HB, CB HN(i+1) and N(i+1) are correct, the peak labels are on top of the real NOE peaks for these atoms and these strips are displayed correctly. However, the remaining strips are from average chemical shift from BMRB and the peak labels are off from peaks, for which we need to find out the correct position. 

<img src="%ATTACHURLPATH%/sc1a.jpg" alt="sc1a.jpg" width='983' height='731' />

#In XEASY, guess the 1H chemical shift for other side-chain groups based on peak pattern from strips [w2(1H),w3(1HN)]-[w2(1H),w3(1HA)]-[w2(1H),w3(1HB)] and BMRB chemical shift for these unassigned groups, start from the group with unique 1H or 13C shift. Adjust the peak position for these unassigned groups, then resort and redisplay the strips. '''Example Figure 2 for Ile 12 side-chain assignment by using NOESY''' This is how it looks after adjusting peak position of QD1, QG2, HG12 and HG13 peak labels to observed peaks that are possibly the real side-chains resonances. This is based on the peak patterns observed in strips of assigned chemical shifts, which need knowledge of peak pattern for different residues. 

<img src="%ATTACHURLPATH%/ile2.jpg" alt="ile2.jpg" width='983' height='731' />

'''Example figure 3 for Ile 12 side-chain assignment by using NOESY''' This is how is looks after strips resort and redisplay, the temporary assigned QD1, QG2, HG12 and HG13 need to be confirmed from the 13C chemical shift.

<img src="%ATTACHURLPATH%/ile3.jpg" alt="ile3.jpg" width='983' height='731' />

#IN XEASY, check strips for unassigned groups from the other view (X-axis: w1(13C/15N), Y-axis:w2(1H); Z-axis: w3(1HC/HN)), check if a corresponding possible 13C shift could be found for the temporary assigned 1H shift.
##Adjust the 13C shift peak position if there is one within the expected 13C shift range and gives an expected peak pattern, resort and redisplay the strips for assignment confirmation. Go to the next unassigned group for this residue. '''Example figure 4 for Ile 12 side-chain assignment by using NOESY''' This is how it looks after adjustment of peak position in 13C dimension. Fortunately, corresponding possible 13C shift are observed for all temporary assigned side-chain resonances QD1, QG2, HG12 and HG13. Adjust 13C chemical shift by adjust peak position in CG2, CG1 and CD1 strips. 

<img src="%ATTACHURLPATH%/ile4.jpg" alt="ile4.jpg" width='983' height='731' />

'''Example figure 5 for Ile 12 side-chain assignment by using NOESY''' This is how it looks after strip resort and redisplay. Now all strips of residue Ile look resonanable and the side-chain assignment of Ile12 is done in simultaneous NOESY and will be confirmed in HCCH.

<img src="%ATTACHURLPATH%/ile5.jpg" alt="ile5.jpg" width='983' height='731' />

##Re-adjust the 1H shift peak position and repeat the previous step search for the corresponding 13C shifts, repeat these steps to get the most reasonable assignment. Or go to the next residue and come back to the side-chain assignment of this residue later.
#Continue on the next residue and repeat these steps until finishing all residues.

Trp e1 and d1 assignment with NOESY

2009-12-18T17:39:20Z

Jlmills: Created page with 'Assignment of the Trp e1 and d1 is easily obtained with the [15N, 1H]-HSQC and simultaneous [1H, 1H]-NOESY '''From b → d1 → e1''' *Prepare the simultaneous [1H, 1H…'

Assignment of the Trp e1 and d1 is easily obtained with the [15N, 1H]-HSQC and simultaneous [1H, 1H]-NOESY

 

'''From b → d1 → e1'''

*Prepare the simultaneous [1H, 1H]-NOESY in [[XEASY|XEASY]] (<tt>ls</tt>, <tt>lc</tt>, <tt>lp</tt>).
*Use <tt>sp</tt> (<tt>sp</tt> and type <tt>TRP</tt> in the box labeled <tt>fragment type</tt> and type <tt>0</tt> in the box labeled <tt>sequential range</tt>), <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to produce strips containing only peaks belonging to Trp residues.
*Start in the strips containing the CB/HB peaks and look for peak around 7ppm. Move the HD1 peak marker vertically to this peak and then update the strip list (<tt>es</tt>, <tt>se</tt>, <tt>gs</tt>)
*Change the orientation of the 3D spectrum to X(13C/15N), Y(1H NOESY), Z(1H detect) using <tt>pm</tt> and then <tt>gs</tt> to display the strips.
*Move the HD1 peak marker horizontally (use <tt>eh</tt>, <tt>gr</tt>, <tt>gl</tt>, and <tt>za</tt> to expand, move, and zoom in as needed) to the correct position. Update the strips (<tt>es</tt>, <tt>se</tt>, <tt>gs</tt>), change back to the standard orientation [<tt>pm</tt>: X(13C/15N), Y(1H NOESY), Z(1H detect)], and display the strips again (<tt>gs</tt>).
*From the newly adjusted HD1 strip, you should see the HE1 peak below (around 10ppm). Move the HE1 peak marker to this position and update the strips (<tt>es</tt>, <tt>se</tt>, <tt>gs</tt>).
*Change the orientation of the 3D spectrum to X(13C/15N), Y(1H NOESY), Z(1H detect) using <tt>pm</tt> and then <tt>gs</tt> to display the strips.
*Move the HE1 peak marker horizontally (use <tt>eh</tt>, <tt>gr</tt>, <tt>gl</tt>, and <tt>za</tt> to expand, move, and zoom in as needed) to the correct position. Update the strips (<tt>es</tt>, <tt>se</tt>, <tt>gs</tt>), change back to the standard orientation [<tt>pm</tt>: X(13C/15N), Y(1H NOESY), Z(1H detect)], and display the strips again (<tt>gs</tt>). 
*Confirm and adjust the positions of HD1 and HE1 if needed.

Resonance Assignment/XEASY

2009-12-18T17:07:12Z

Jlmills:

#[[XEASY Introduction|Introduction]]
#[[XEASY Spin system identification|Spin system identification]]
#Backbone resonance assignment''' '''
##GFT-based spectra
###[[HNCACAB/CABCA(CO)NH]]
##Conventional spectra
###[[HNCACB/CBCA(CO)NH]]
#Side chain resonance assignment
##Aliphatic
###GFT-based spectra
####[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
####[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
###Conventional spectra
####HAHB(CO)NH
####HCCH-COSY
####[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
##[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
##Additional side chain atoms
###[[Trp_e1_and_d1_assignment_with_NOESY|Trp e1 NH and d1 CH]]
###[[Met methyl assignment with NOESY|Met e CH3 ]]
###[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#NOESY peak integration

Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

2009-12-18T17:05:17Z

Jlmills:

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|aliphatic (4,3)D HCCH Analysis]] for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

'''Phe, Tyr, and His'''

*Run the following macro in UBNMR to generate a peaklist and atom list: 
<pre> init
read seq myprot.seq #change filename to something appropriate
read prot noe.prot #change filename to your most current prot list

add GFTatom COSY_C ATTACHED_H

simulate 3D CG COSY_H 0 1
simulate 3D CG COSY_C 0 1
simulate 3D CG1 COSY_CH 0 1
simulate 3D CG1 COSY_C 0 1
simulate 3D CG2 COSY_CH 0 1
simulate 3D CG2 COSY_C 0 1

simulate 3D CD COSY_CH 0 2
simulate 3D CD COSY_C 0 2
simulate 3D CD1 COSY_CH 0 2
simulate 3D CD1 COSY_C 0 2
simulate 3D CD2 COSY_CH 0 2
simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
simulate 3D CZ2 COSY_C 0 3
simulate 3D CZ3 COSY_CH 0 3
simulate 3D CZ3 COSY_C 0 3
simulate 3D CE3 COSY_CH 0 4
simulate 3D CE3 COSY_C 0 4
simulate 3D CH2 COSY_CH 0 4
simulate 3D CH2 COSY_C 0 4

write prot hcchAroI1.prot #the newly created starting prot list
write peaks hcchAroI1.peaks #the newly created starting peak list
</pre>
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips.
*In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts
*use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList.
*In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat the above steps for the QE<tt>strips</tt> and assign QE. Note that for His, the assignment is often complicated by the presence of strong signals from the His-tag introduced to facilitate protein purification. The QE strip in the basic spectra is expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possibly 13CZ±1HZ (for Phe only).

'''TRP'''

*In XEASY, repeat steps 1-3 for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3.

Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

2009-12-18T17:04:54Z

Jlmills:

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|aliphatic (4,3)D HCCH Analysis]] for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

'''Phe, Tyr, and His'''

*Run the following macro in UBNMR to generate a peaklist and atom list: 
<pre> init
read seq myprot.seq #change filename to something appropriate
read prot noe.prot #change filename to your most current prot list

add GFTatom COSY_C ATTACHED_H

simulate 3D CG COSY_H 0 1
simulate 3D CG COSY_C 0 1
simulate 3D CG1 COSY_CH 0 1
simulate 3D CG1 COSY_C 0 1
simulate 3D CG2 COSY_CH 0 1
simulate 3D CG2 COSY_C 0 1

simulate 3D CD COSY_CH 0 2
simulate 3D CD COSY_C 0 2
simulate 3D CD1 COSY_CH 0 2
simulate 3D CD1 COSY_C 0 2
simulate 3D CD2 COSY_CH 0 2
simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
simulate 3D CZ2 COSY_C 0 3
simulate 3D CZ3 COSY_CH 0 3
simulate 3D CZ3 COSY_C 0 3
simulate 3D CE3 COSY_CH 0 4
simulate 3D CE3 COSY_C 0 4
simulate 3D CH2 COSY_CH 0 4
simulate 3D CH2 COSY_C 0 4

write prot hcchAroI1.prot #the newly created starting prot list
write peaks hcchAroI1.peaks #the newly created starting peak list
</pre>
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips.
*In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts
*use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList.
*In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat the above steps for the QE<tt>strips</tt> and assign QE. Note that for His, the assignment is often complicated by the presence of strong signals from the His-tag introduced to facilitate protein purification. The QE strip in the basic spectra is expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possibly 13CZ±1HZ (for Phe only).[[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Side chain assignments using simultaneous 15N]]

'''TRP'''

*In XEASY, repeat steps 1-3 for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3.

Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

2009-12-18T17:04:02Z

Jlmills:

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[Side_chain_assignment_with_aliphatic_(4,3)D_HCCH-COSY_in_XEASY|aliphatic (4,3)D HCCH Analysis]] for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

'''Phe, Tyr, and His'''

*Run the following macro in UBNMR to generate a peaklist and atom list: 
<pre> init
read seq myprot.seq #change filename to something appropriate
read prot noe.prot #change filename to your most current prot list

add GFTatom COSY_C ATTACHED_H

simulate 3D CG COSY_H 0 1
simulate 3D CG COSY_C 0 1
simulate 3D CG1 COSY_CH 0 1
simulate 3D CG1 COSY_C 0 1
simulate 3D CG2 COSY_CH 0 1
simulate 3D CG2 COSY_C 0 1

simulate 3D CD COSY_CH 0 2
simulate 3D CD COSY_C 0 2
simulate 3D CD1 COSY_CH 0 2
simulate 3D CD1 COSY_C 0 2
simulate 3D CD2 COSY_CH 0 2
simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
simulate 3D CZ2 COSY_C 0 3
simulate 3D CZ3 COSY_CH 0 3
simulate 3D CZ3 COSY_C 0 3
simulate 3D CE3 COSY_CH 0 4
simulate 3D CE3 COSY_C 0 4
simulate 3D CH2 COSY_CH 0 4
simulate 3D CH2 COSY_C 0 4

write prot hcchAroI1.prot #the newly created starting prot list
write peaks hcchAroI1.peaks #the newly created starting peak list
</pre>
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips.
*In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts
*use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList.
*In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat the above steps for the QE<tt>strips</tt> and assign QE. Note that for His, the assignment is often complicated by the presence of strong signals from the His-tag introduced to facilitate protein purification. The QE strip in the basic spectra is expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possibly 13CZ±1HZ (for Phe only). .See also: [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]]

'''TRP'''

*In XEASY, repeat steps 1-3 for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3. See also [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]]

Aromatic side chain assignment with Aro-HCCH-COSY in XEASY

2009-12-18T16:37:58Z

Jlmills:

Aromatic (4,3)D HCCH shows signals for aromatic H-C-C-H moeties. See [[NESG:Aliphatic side-chain resonance assignment using the aliphatic HCCH COSY spectrum|aliphatic (4,3)D HCCH Analysis]] for comparison.

Before proceeding, it is helpful to look for the QD (Phe and Tyr), HD2 (His), and HD1 (Trp) resonances in the simultaneous [1H, 1H]-NOESY. Look for a consistent peak that is visible in the amide, alpha, and beta strips near 7ppm.

'''Phe, Tyr, and His'''

*Run the following macro in UBNMR to generate a peaklist and atom list: 
<pre> init
read seq myprot.seq #change filename to something appropriate
read prot noe.prot #change filename to your most current prot list

add GFTatom COSY_C ATTACHED_H

simulate 3D CG COSY_H 0 1
simulate 3D CG COSY_C 0 1
simulate 3D CG1 COSY_CH 0 1
simulate 3D CG1 COSY_C 0 1
simulate 3D CG2 COSY_CH 0 1
simulate 3D CG2 COSY_C 0 1

simulate 3D CD COSY_CH 0 2
simulate 3D CD COSY_C 0 2
simulate 3D CD1 COSY_CH 0 2
simulate 3D CD1 COSY_C 0 2
simulate 3D CD2 COSY_CH 0 2
simulate 3D CD2 COSY_C 0 2

simulate 3D CZ2 COSY_CH 0 3
simulate 3D CZ2 COSY_C 0 3
simulate 3D CZ3 COSY_CH 0 3
simulate 3D CZ3 COSY_C 0 3
simulate 3D CE3 COSY_CH 0 4
simulate 3D CE3 COSY_C 0 4
simulate 3D CH2 COSY_CH 0 4
simulate 3D CH2 COSY_C 0 4

write prot hcchAroI1.prot #the newly created starting prot list
write peaks hcchAroI1.peaks #the newly created starting peak list
</pre>
*In XEASY, use <tt>ns</tt> to load the three sub-spectra of aromatic (4,3)D HCCH; use <tt>ls</tt>, <tt>lp</tt> and <tt>lc</tt> to load the sequence, peak list, and chemical shift list, respectively and <tt>se</tt>, <tt>gs</tt> to sort and display [w1(13C;1H);w3(1H)]-strips. In XEASY, use <tt>pm</tt>, <tt>es</tt>, <tt>se</tt> and <tt>gs</tt> (or <tt>sf</tt>) to display [w1(13C;1H);w2(13C)]-planes, and sort and [w1(13C;1H);w2(13CD)]-planes; use <tt>mr</tt> to accurately adjust peak positions to assign 13CD chemical shifts; use <tt>pm</tt> and <tt>gs</tt> to re-display [w1(13C;1H); w3(1H)]-planes and [w1(13C;1H); w3(1H)]-strips; use <tt>mr</tt> to accurately adjust peak positions to confirm QD chemical shifts. The strips in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, ..., 13CD±QD and 13CD±QE. Use <tt>mr</tt> to accurately position peaks along w1; use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save updated PeakList and AtomList. In UBNMR, run <tt>updatacosyGFT</tt> to calculate SQ shifts from updated AtomList. Repeat 1.- 3. and 4. for <tt>QE strips</tt> and assign HE. Note that for His, assignment is often complicated by the presence of strong signals from HIS-Tags introduced to facilitate protein purification. The QE strip in the basic spectra are expected to exhibit peaks at 13CD, 13Main.CE, 13CZ, ..., 13CD±QD, 13Main.CE±QE and possible 13CZ±1HZ (for Phe only). .See also: [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]].

*'''TRP'''

##In XEASY, for residue Trp, repeat steps 1.-3. for strips in the order HH2 > HZ2 > HZ3 > HE3 (instead of the strip order QD > QE for Tyr and Phe). In HH2-strips, assign 13CH2±1HH2, and 13CZ2±1HZ2. In HZ2-strips assign 13CZ2± 1HH2, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3-strips, assign 13CZ3±1HZ3, 13CZ2±1HZ2 and 13CZ3±1HZ3. In HZ3 strip, assign 13CZ3±1HZ3, and 13CE3±1HE3. See also [[NESG:Side chain assignments using simultaneous 15N, 13C-resolved (1H,1H) NOESY|Assign Aromatic Side-chain Resonances using the NOESY and HCCH]].
##In XEASY, use <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to save the final PeakList and AtomList

-- Main.GaohuaLiu - 01 Feb 2007

Met methyl assignment with NOESY

2009-12-18T16:07:20Z

Jlmills:

The Met methyl group has unique chemical shifts with 1H around 2.0ppm and 13C at 17ppm. Therefore, the best way to identify Met methyl is starting from the aliphatic [13C, 1H]-HSQC. As most proteins have very few methionines, the assignment strategy described below takes a "manual" approach. It is also recommended to obtain aliphatic side-chain assignment before pursuing the following approach. 

#Open two instances of XEASY: one with the aliphatic [13C, 1H]-HSQC and the other with the simultaneous [1H, 1H]-NOESY. Prepare the NOESY by loading the sequence file, atom list, and peak list. Select only the methionine residues (<tt>sp</tt> and type <tt>MET</tt> in the box labeled <tt>fragment type</tt> and type 0<tt></tt> in the box labeled <tt>sequential range</tt>) and create the strips. 
#Go to the HSQC and look for the methionine methyl groups. They should be isolated from most other peaks (around 2ppm in 1H and 17ppm in 13C) and their sign will be opposite of the other methyl peaks in a 28ms constant time HSQC. Note the chemical shifts for each peak.
#Go back to the NOESY and look at all of the strips for residue "X". Place the cursor in the last strip of residue "X" and type <tt>zd</tt> (zoom duplicate) to create another strip. Place the cursor in the new strip and type <tt>gp</tt> (go to plane). In the pop-up dialog window, type the 13C chemical shift of one of the methyl groups. (NOTE: it is important to type the chemical shift in as "17.65p" to indicate ppm. If you type only "17.65", then you will go to plane 17.) Next, use <tt>gr</tt> and <tt>gl</tt> to move the center of the strip to the correct 1H chemical shift.
#Compare the peak patterns to determine if the current Met methyl peak corresponds to residue "X". If it does, pick the peak (<tt>pp</tt>) and assign it (<tt>ap</tt>). If it does not, go back to step 3 and repeat using the chemical shifts of a different Met methyl from the HSQC.

Amide Side Chain assignment with NOESY

2009-12-18T15:41:50Z

Jlmills:

This section describes the procedure to assign the amide side chains of Asn (d2) and Gln (e2) using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY (or by combining separate 15N resolved and 13C-resolved [1H, 1H]-NOESY spectra). Assuming that you have complete assignments of the Asn/Gln aliphatic side chain, extending the assignment to include the d2 and e2 is straightforward.

 

#Load two copies of the NOESY into XEASY. Be sure to use two different orientations for the two spectra. Spectrum I should be viewed as 13C/15N(x-axis), indirect 1H(y-axis), and direct 1H(z-axis) while spectrum II should be viewed as direct 1H(x-axis), indirect 1H(y-axis), and 13C/15N (z-axis). Additionally, load the appropriate peak list, sequence list, and atom list. Select Asn and Gln peaks with <tt>sp</tt> (type <tt>ASN GLN</tt> in the box labeled <tt>fragment type</tt> and type <tt>0</tt> in the box labeled <tt>sequential range</tt>). Create strips in both spectra (<tt>es</tt>, <tt>se</tt>, <tt>rc</tt>, <tt>rs</tt>, <tt>se</tt>, <tt>gs</tt>).
#Look at the CB strips of Asn or the CG strips of Gln. You should see 2 strong cross peaks near 7ppm in both the HB2(HG2) and HB3(HG3) strips which correspond to HB-HD21/HB-HD22 or HG-HE21/HG-HE22. At this point, it is helpful to create 4 new peaks (ND2/HD22/HD22, ND2, HD21/HD21, ND2/HD22/HD21, and ND2/HD21/HD22 for Asn and NE2/HE22/HE22, NE2, HE21/HE21, NE2/HE22/HE21, and NE2/HE21/HE22 for Gln. The location of these peaks is not important at this time because they will be moved to the proper location.) Once you have identified the 2 amide side chain peaks and created the new peaks, use <tt>mr</tt> to move the HD22/HD21 (or HE22/HE21) peaks in spectrum II (the one with the direct 1H dimension along the x-axis). 
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to see the updated peak positions and the newly created peaks. Use <tt>mr</tt> again to move the peaks in spectrum I. 
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to verify the new assignments.

Amide Side Chain assignment with NOESY

2009-12-10T23:42:09Z

Jlmills:

== '''Amide Side chain resonance assignment using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY''' ==

This section describes the procedure to assign the amide side chains of Asn (d2) and Gln (e2) using the simultaneous 15N, 13C-resolved [1H, 1H]-NOESY (or by combining separate 15N resolved and 13C-resolved [1H, 1H]-NOESY spectra). Assuming that you have complete assignments of the Asn/Gln aliphatic side chain, extending the assignment to include the d2 and e2 is straightforward.

 

#Load two copies of the NOESY into XEASY. Be sure to use two different orientations for the two spectra. Spectrum I should be viewed as 13C/15N(x-axis), indirect 1H(y-axis), and direct 1H(z-axis) while spectrum II should be viewed as direct 1H(x-axis), indirect 1H(y-axis), and 13C/15N (z-axis). Additionally, load the appropriate peak list, sequence list, and atom list. Select Asn and Gln peaks with <tt>sp</tt> (type <tt>ASN GLN</tt> in the box labeled <tt>fragment type</tt> and type <tt>0</tt> in the box labeled <tt>sequential range</tt>). Create strips in both spectra (<tt>es</tt>, <tt>se</tt>, <tt>rc</tt>, <tt>rs</tt>, <tt>se</tt>, <tt>gs</tt>).
#Look at the CB strips of Asn or the CG strips of Gln. You should see 2 strong cross peaks near 7ppm in both the HB2(HG2) and HB3(HG3) strips which correspond to HB-HD21/HB-HD22 or HG-HE21/HG-HE22. At this point, it is helpful to create 4 new peaks (ND2/HD22/HD22, ND2, HD21/HD21, ND2/HD22/HD21, and ND2/HD21/HD22 for Asn and NE2/HE22/HE22, NE2, HE21/HE21, NE2/HE22/HE21, and NE2/HE21/HE22 for Gln. The location of these peaks is not important at this time because they will be moved to the proper location.) Once you have identified the 2 amide side chain peaks and created the new peaks, use <tt>mr</tt> to move the HD22/HD21 (or HE22/HE21) peaks in spectrum II (the one with the direct 1H dimension along the x-axis). 
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to see the updated peak positions and the newly created peaks. Use <tt>mr</tt> again to move the peaks in spectrum I. 
#Recreate the strips (<tt>es</tt>, <tt>se</tt>, <tt>se</tt>, <tt>gs</tt>) to verify the new assignments.

Amide Side Chain assignment with NOESY

2009-12-10T23:40:32Z

Jlmills:

Resonance Assignment/XEASY

2009-12-10T22:44:36Z

Jlmills:

#[[XEASY Introduction|Introduction]]
#[[XEASY Spin system identification|Spin system identification]]
#Backbone resonance assignment''' '''
##GFT-based spectra
###[[HNCACAB/CABCA(CO)NH]]
##Conventional spectra
###[[HNCACB/CBCA(CO)NH]]
#Side chain resonance assignment
##Aliphatic
###GFT-based spectra
####[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
####[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
###Conventional spectra
####HAHB(CO)NH
####HCCH-COSY
####[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
##[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
##Additional side chain atoms
###Trp e1 NH and d1 CH
###[[Met methyl assignment with NOESY|Met e CH3 ]]
###[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#NOESY peak integration

Processing NMR spectra with UB PERL scripts

2009-12-07T16:16:30Z

Jlmills: Created page with '= Semi-automated processing of NMR spectra with PERL scripts developed at UB = == Introduction =='

= Semi-automated processing of NMR spectra with PERL scripts developed at UB =

== Introduction ==

File:HCAL.txt

2009-12-04T21:39:45Z

Jlmills:

File:Experimentlist.txt

2009-12-04T21:39:30Z

Jlmills:

Wiki Tree Layout

2009-12-04T20:07:59Z

Jlmills:

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

*It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
*There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
*We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
*"Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
*Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page

 

= HTP NMR structure determination =

== Protein Target Selection, Sample Preparation, and Initial Screening ==

#[[Target selection|NESG target selection]] 
#[[DNA cloning protocols|DNA cloning protocols]] 
#[[Protein purification|Protein expression and purification protocols]]  
#Sample Optimization
##[[Construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##[[NMR screening]]
 

== NMR Data Collection ==

#Routine operation
##[[NMR sample tubes]]
##[[NMR Sample Preparation]]
##[[Inserting NMR Sample]]
##Tuning and matching
##[[Deuterium Lock]]
##[[Shimming]]
##[[Pulse width calibration]]
##[[Temperature calibration]]
##[[Chemical shift referencing]]
#Advanced operation
##[[Deuterium pulse width calibration and decoupling]]
#NMR data acquisition for protein structure determination
##[[Common NMR experiment sets]]
##[[NMR experiment setup scripts for VNMRJ|Custom NMR experiment setup scripts for VNMRJ]]
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC
##[[Estimation of rotational correlation time]]
##[[Estimation of measurement time]]
##NMR experiments for spin system identification
##2D and 3D NOESY experiments
###[[Simultaneous 13C,15N-resolved NOESY]]
##Double and triple NMR experiments
###3D CBCA(CO)NH and HNCACB
###3D HNCA and HN(CO)CA
###3D HAHB(CO)NH
###(4,3)D CABCA(CO)NH and HNCACB
###(4,3)D HABCAB(CO)NH
###(H)CCH
###(H)CCH-TOCSY
###H(C)CH
###H(C)CH-TOCSY
###(4,3)D HCCH
##Other NMR experiments
###[[2D (13C, 1H) HSQC for fractionally 13C-labeled samples|2D [13C, 1H]-HSQC for fractionally 13C-labeled samples]]
###[[Long-range 15N-1H correlation experiments for histidine rings]] - determination of histidine isoprotomer state
###MEXICO
###CLEANEX
###H-D exchange experiment
###15N spin relaxation parameters
#Advanced problems for data collection
##[[Setting up non-uniformly sampled spectra]]
###[[Setting up non-uniformly sampled spectra/NUS guide for Varian|Guide for Varian/BioPack]]
###[[Setting up non-uniformly sampled spectra/NUS guide for Bruker according to Arrowsmith group in Toronto|Guide for Bruker/Topspin according to Arrowsmith group]]
#Maintenance
##VARIAN
###[[Installing and updating BioPack]]
###[[Full probefile calibration]]
###[[Rebooting spectrometer console]]
###[[Conditioning procedure for cryogenic probes]]
##BRUKER

== NMR Data Processing ==

#Processing Procedures for Routine Experiments 
##NMRPipe
###[[Brief description of philosophy, commands, and functions of NMRPipe|Brief description of philosophy, commands, and functions of NMRPipe]]
###[[Routine 2D Experiment|Routine Processing Procedure for 2D Experiment]]
###[[Routine Processing Procedure for 3D 15N and 13C-edited Experiments|Routine Processing Procedure for 3D 15N and 13C-edited Experiments]]
###[[HSQCTROSY RDC Measurement|2D ]][[HSQCTROSY RDC Measurement|15]][[HSQCTROSY RDC Measurement|N HSQC-TROSY experiment for RDC measurement]]
###[[Jmodulation Experiment RDC|2D J-modulation experiment for RDC measurement]]
###[[Processing_NMR_spectra_with_UB_PERL_scripts|Routine processing of spectra using Buffalo-developed PERL scripts]]
##[[Processing NMR spectra with PROSA|PROSA]]
##TOPSPIN
##[[AGNuS/AutoProc|AUTOPROC]]
##[[UBNMR|UBNMR]]
#Processing Procedures for Alternative Data Collection Methods
##G-Matrix Fourier transformation (GFT)
##[[Processing non-uniformly sampled spectra with Multidimensional Decomposition|Processing non-uniformly sampled spectra with Multidimensional Decomposition]]
#Spectra Format Conversion
##NMRPipe processed data conversion to Sparky, CARA, XEASY, and NMRViewJ
##TOPSPIN processed data conversion to Sparky, CARA, XEASY, and NMRViewJ 

== Resonance Assignment ==

This chapter focuses on data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.

 

#[[Resonance Assignment/Principles and concepts|Principles and concepts]]
#[[Resonance Assignment/Practical aspects|Practical aspects]]
##Semi-automated protocols
###[[Resonance Assignment/CARA|CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####Side Chain Assignment
#####[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
#####[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
#####[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
###[[Sparky]]
###[[Resonance Assignment/XEASY|XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####Backbone resonance assignment''' '''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####Side chain resonance assignment
#####Aliphatic
######GFT-based spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|Assignment validation suite (AVS)]]
###[[LACS|Linear analysis of chemical shift (LACS)]]
##[[PDB and BMRB Deposition#Preparing_files_for_BMRB_depostion|Depositing chemical shifts]]

 

== Structure Calculation and Validation ==

#[[Structure Calculation and Validation|Principles and concepts]]
#Practical aspects
##Structure calculation
###CYANA
####[[CYANA|Getting started]]
####[[FOUND|FOUND]]
####[[TALOS|TALOS]]
####[[GLOMSA|GLOMSA]]
####[[NOE Calibration Using CYANA|NOE calibration]]
####[[Manual Structure Calculation Using CYANA|Manual structure calculation]]
####[[Automated NOESY Assignment Using CYANA|Automated NOESY assignment and structure calculation]]
####[[Structure Calculation With RDC's Using CYANA|Structure calculation with residual dipolar couplings]] (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA| ]]
###AutoStructure
####[[AutoStructure|Getting started]]
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]
####[[XPLOR Structure Calculations Using AutoStructure|XPLOR run]]
####[[Analyzing AutoStructure Output Directories|Analyzing the output]]
####[[RPF Analysis|RPF/DP scores]]
####[[Structure Calculation Using AS-DP|Structure calculation using AS-DP]]
###"Consensus" Approaches
####[[Overview of Consensus Runs|Overview of Consensus runs]]
####[[Finding Consensus NOE Assignments|Finding Consensus NOE assignments]]
####[[Validation of Consensus Run|Validation of Consensus runs]]
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
###[[Structure Calculation Using CS-DP ROSETTA|CS-DP ROSETTA]]
###[[Structure Calculation Using CS-RDC-ROSETTA|CS-RDC-ROSETTA]]
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]] 
###Special topics
####[[Protein-Ligand Complexes|Protein-Ligand complexes]]
####[[Working With Metal Ions|Metal ions]]
####[[Working With Dimers|Dimers]]
####[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
####[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
####[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
##Structure Refinement
###[[Structure Refinement Using CNS Energy Minimization With Explicit Water|CNS refinement]]
###[[Structure Refinement Using XPLOR-NIH|XPLOR-NIH refinement]]
###[[Rosetta High Resolution Protein Structure Refinement Protocol|ROSETTA refinement]]
##Validation and deposition
###[[PdbStat|PdbStat]]
###[[PSVS|PSVS]]
###[[RPF Analysis|RPF analysis]]
###[[MolProbity Server|MolProbity server]]
###[[PDB and BMRB Deposition|PDB and BMRB deposition]]
###[[ADIT-NMR|ADIT-NMR]]
###[[HarvestDB|HarvestDB]]
###[[SPINS|SPINS]]

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009

UBNMR

2009-12-03T17:24:21Z

Jlmills:

== '''UBNMR''' ==

Please click to download files:

*[[Media:UBNMR-manua-v3_ts_(2).doc|UBNMR-manua-v3_ts_(2).doc]]: UBNMR Manual
*[[Media:UBNMR_1.3.1.tar.gz|UBNMR_1.3.1.tar.gz]]: UBNMR version 1.3.1
*[[Media:UbnmrInstall.doc|UbnmrInstall.doc]]: UBNMR installation instruction

Please contact [mailto:jlmills@buffalo.edu Jeff] for addtional assistance on UBNMR.

 -- AlexEletski - 16 Jan 2007

-- Updated by JimAramini - Nov 2009

HA and HB Assignment with GFT in XEASY

2009-12-01T18:07:36Z

Jlmills:

== '''HA And HB Assignments with XEASY/UBNMR''' ==

HA and HB assignments provide the bridge from [[XEASY Backbone Assignment|Backbone Assignment]] to the complete [[XEASY Side Chain Assignments|Side Chain Assignments]].

=== '''Analysis of the(4,3)D GFT HABCAB(CO)NHN''' ===

The sequence-specific 15N / 1HN / 13CAB resonance assignments obtained as described in backbone assignment are first used to also obtain 1HA and 1HB shift assignments by analyzing (4,3)D HABCAB(CO)NHN. Then, shifts of more peripheral aliphatic spins are obtained from (4,3)D HCCH.

#Go to the directory <tt>/analisys/xeasy/habcab</tt> and copy the <tt>final-clean.seq</tt> and <tt>final-clean.prot</tt> files from the backbone directory
#Run <tt>makeHabcabPeaks</tt> in [[UBNMR|UBNMR]] to generate an extended [[XEASY Atom List|AtomList]] <tt>habcabconhI1.prot</tt> (containing linear combinations of 13CAB and 1HAB shifts) and the initial HABCAB [[XEASY Peak List|PeakList]] <tt>habcabconhI1.peaks</tt> to guide manual peak identification in (4,3)D HABCAB(CO)NHN. Peaks are colored according to type of sub-spectrum and type of proton involved in the GFT dimension.
#In XEASY
##use <tt>ns</tt> to load '''subspectrum I''', <tt>HABCABcoNH1</tt>, from <tt>/protName/analysis/xeasy/habcab/</tt>
##<tt>cp</tt> to display the spectrum as a contour plot
##<tt>ls</tt> to load the [[XEASY Sequence List|SequencList]] (<tt>protein.seq</tt>)
##<tt>lc</tt> to load [[XEASY Atom List|AtomList]] (<tt>habcabconhI1.prot</tt>)
##<tt>lp</tt> to load the starting [[XEASY Peak List|PeakList]] (<tt>habcabconhI1.peaks</tt>)
##<tt>se</tt> to create the strips for '''subspectrum I'''
##<tt>ns</tt> to load '''subspectrum II''', <tt>HABCABcoNH2</tt>
##<tt>cp</tt> to display the spectrum as a contour plot
##<tt>se</tt> to create the strips for '''subspectrum II''' and <tt>gs</tt> to display the strips (see Figure 1, below)
##<tt>pw</tt> and click the box for '''assignment y''' to display the assignments in the GFT HabCab dimension
##<tt>mr</tt> to accurately position the peaks along w1(13C) and <tt>bs</tt> / <tt>fs</tt> to move through the strips
###when moving peaks, be sure to move the proper peak for each of the subspectra ('''NOTE''': if the spectra were processed using Buffalo's PERL scripts, then '''subspectrum I''' should use the "plus" peaks -- CA+HA, CB+HB2, CB+HB3, etc.)
###if the HA1/2 or HB2/3 peaks are degenerate, move both of the peak markers to the same position and try to resolve them in the NOESY 
###for non-degenerate HB2/3 (or HA1/2) peaks, place the CB+HB3 and CB-HB3 peaks on the "outside" and the CB+HB2 and CB-HB2 peaks on the "inside" of the peak pairs
###if you are unable to correctly identify the peak pairs (e.g., CA+HA and CA-HA), use the 13C single-quantum chemical shifts from the backbone experiments to identify the center of the peak pair
#In UBNMR, run <tt>updateHabAtom</tt> to update single-quantum 1HAB and 13CAB shifts as <tt>habcabconhO2.prot</tt>. A least-squares fit provides the single-quantum HA/HB chemical shifts (consistency of peaks representing different linear combinations of shifts is checked by UBNMR in oder to identify inadvertently 'mis-picked' peaks; a warning is then provided).
#In XEASY, double-check the peak positions for residues that gaving large error. Repeat previous step and this step till no improvement can be made.
#Now it is ready to move to [[XEASY Side Chain Assignments|Side Chain Assignments]]
#'''Figure 1: Example of (4,3)D GFT HABCAB(CO)NHN analysis before (left) and after (right) peak position adjustment by <tt>mr</tt>.''' [[Image:XEASY hab5.jpg]] [[Image:XEASY hab4.jpg]] 

 

 

*[[Media:XEASY_makeHabcabPeaks.txt|makeHabcabPeaks]]: UBNMR macro

*[[Media:XEASY_updateHabAtom.txt|updateHabAtom]]: UBNMR macro

HA and HB Assignment with GFT in XEASY

2009-12-01T17:08:16Z

Jlmills:

== '''HA And HB Assignments with XEASY/UBNMR''' ==

HA and HB assignments provide the bridge from [[XEASY Backbone Assignment|Backbone Assignment]] to the complete [[XEASY Side Chain Assignments|Side Chain Assignments]].

=== '''Analysis of the(4,3)D GFT HABCAB(CO)NHN''' ===

The sequence-specific 15N / 1HN / 13CAB resonance assignments obtained as described in backbone assignment are first used to also obtain 1HA and 1HB shift assignments by analyzing (4,3)D HABCAB(CO)NHN. Then, shifts of more peripheral aliphatic spins are obtained from (4,3)D HCCH.

#Go to the directory <tt>/analisys/xeasy/habcab</tt> and copy the <tt>final-clean.seq</tt> and <tt>final-clean.prot</tt> files from the backbone directory
#Run <tt>makeHabcabPeaks</tt> in [[UBNMR|UBNMR]] to generate an extended [[XEASY Atom List|AtomList]] <tt>habcabconhI1.prot</tt> (containing linear combinations of 13CAB and 1HAB shifts) and the initial HABCAB [[XEASY Peak List|PeakList]] <tt>habcabconhI1.peaks</tt> to guide manual peak identification in (4,3)D HABCAB(CO)NHN. Peaks are colored according to type of sub-spectrum and type of proton involved in the GFT dimension.
#In XEASY
##use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt> and <tt>lp</tt> to load the sub-spectra of (4,3)D HABCAB(CO)NHN and the starting HABCAB-peak list, then use <tt>se</tt>, <tt>gs</tt> to display [w1(13C,1H),w3(1HN)]-strips and use <tt>mr</tt> to accurately position peaks
#Use <tt>aa</tt>, <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to update the peak list as <tt>habcabconhO1.peaks</tt> and AtomList as <tt>habcabconhO1.prot</tt>. See the analysis procedure below.
#In XEASY, use <tt>ns</tt> to load subspectrum I, <tt>HABCABcoNH1</tt>, from <tt>/protName/analysis/xeasy/data/</tt>; <tt>ls</tt> to load the sequence file, e.g. <tt>protein.seq</tt>; <tt>lc</tt> to load AtomList from <tt>habcabconhI1.prot</tt>; <tt>lp</tt> to load the starting peaklist <tt>habcabconhI1.peaks</tt>; use <tt>ns</tt> to load sub-spectrum II, <tt>HABCABcoNH2</tt>.

#In XEASY, use <tt>cp</tt> to display the spectrum as a countour plot; use <tt>fp</tt> or <tt>bp</tt> to step through the planes until you see representative peaks for setting the coutour levels; use <tt>pm</tt> to display [w1(13C/1H),w2(15N)]-planes; use <tt>mr</tt> to accurately position the peaks along w2(15N); use <tt>pm</tt> to display [w2(15N),w3(1HN)]-planes.
#In XEASY, use <tt>se</tt> and <tt>gs</tt> to create strips from entries of loaded PeakList and display, for example, ten at once; navigate through the strips by using <tt>fs</tt> and <tt>bs</tt>; use <tt>pw</tt> to set the '''assignment y''' in the peak window so that only the assignment in the GFT dimension is displayed. One should see strips from both sub-spectra assigned to the same residue side-by-side.
#In XEASY, use <tt>gs</tt>, <tt>sf</tt>, <tt>fs</tt> and <tt>bs</tt> to go through strips and use <tt>mr</tt> to move the peaks to the actual peak positions. Make sure that the peaks are moved in the right sub-spectrum. If two HB-proton shifts are apparently degenerate, move the corresponding peak to the same position (note that in the better resolved NOESY, the two shifts may turn out to be non-degenerate). If the assignment is ambiguous, load 'central peak spectrum' to identify peak pairs in the sub-spectra. '''Figure 1: Example of (4,3)D GFT HABCAB(CO)NHN analysis before (left) and after (right) peak position adjustment by <tt>mr</tt>.''' [[Image:XEASY hab5.jpg]] [[Image:XEASY hab4.jpg]] 
#In UBNMR, run <tt>updateHabAtom</tt> to update single-quantum 1HAB and 13CAB shifts as <tt>habcabconhO2.prot</tt>. A least-squares fit provides the single-quantum HA/HB chemical shifts (consistency of peaks representing different linear combinations of shifts is checked by UBNMR in oder to identify inadvertently 'mis-picked' peaks; a warning is then provided).
#In XEASY, double-check the peak positions for residues that gaving large error. Repeat previous step and this step till no improvement can be made.
#Now it is ready to move to [[XEASY Side Chain Assignments|Side Chain Assignments]]

 

 

*[[Media:XEASY_makeHabcabPeaks.txt|makeHabcabPeaks]]: UBNMR macro

*[[Media:XEASY_updateHabAtom.txt|updateHabAtom]]: UBNMR macro

HA and HB Assignment with GFT in XEASY

2009-12-01T17:01:18Z

Jlmills:

== '''HA And HB Assignments with XEASY/UBNMR''' ==

HA and HB assignments provide the bridge from [[XEASY Backbone Assignment|Backbone Assignment]] to the complete [[XEASY Side Chain Assignments|Side Chain Assignments]].

=== '''Analysis of the(4,3)D GFT HABCAB(CO)NHN''' ===

The sequence-specific 15N / 1HN / 13CAB resonance assignments obtained as described in backbone assignment are first used to also obtain 1HA and 1HB shift assignments by analyzing (4,3)D HABCAB(CO)NHN. Then, shifts of more peripheral aliphatic spins are obtained from (4,3)D HCCH.

#Go to the directory <tt>/analisys/xeasy/habcab</tt> and copy the <tt>final-clean.seq</tt> and <tt>final-clean.prot</tt> files from the backbone directory
#Run <tt>makeHabcabPeaks</tt> in [[UBNMR|UBNMR]] to generate an extended [[XEASY Atom List|AtomList]] <tt>habcabconhI1.prot</tt> (containing linear combinations of 13CAB and 1HAB shifts) and the initial HABCAB [[XEASY Peak List|PeakList]] <tt>habcabconhI1.peaks</tt> to guide manual peak identification in (4,3)D HABCAB(CO)NHN. Peaks are colored according to type of sub-spectrum and type of proton involved in the GFT dimension.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt> and <tt>lp</tt> to load the sub-spectra of (4,3)D HABCAB(CO)NHN and the starting HABCAB-peak list, then use <tt>se</tt>, <tt>gs</tt> to display [w1(13C,1H),w3(1HN)]-strips and use <tt>mr</tt> to accurately position peaks
#Use <tt>aa</tt>, <tt>ac</tt>, <tt>wp</tt> and <tt>wc</tt> to update the peak list as <tt>habcabconhO1.peaks</tt> and AtomList as <tt>habcabconhO1.prot</tt>. See the analysis procedure below.
#In XEASY, use <tt>ns</tt> to load subspectrum I, <tt>HABCABcoNH1</tt>, from <tt>/protName/analysis/xeasy/data/</tt>; <tt>ls</tt> to load the sequence file, e.g. <tt>protein.seq</tt>; <tt>lc</tt> to load AtomList from <tt>habcabconhI1.prot</tt>; <tt>lp</tt> to load the starting peaklist <tt>habcabconhI1.peaks</tt>; use <tt>ns</tt> to load sub-spectrum II, <tt>HABCABcoNH2</tt>.

#In XEASY, use <tt>cp</tt> to display the spectrum as a countour plot; use <tt>fp</tt> or <tt>bp</tt> to step through the planes until you see representative peaks for setting the coutour levels; use <tt>pm</tt> to display [w1(13C/1H),w2(15N)]-planes; use <tt>mr</tt> to accurately position the peaks along w2(15N); use <tt>pm</tt> to display [w2(15N),w3(1HN)]-planes.
#In XEASY, use <tt>se</tt> and <tt>gs</tt> to create strips from entries of loaded PeakList and display, for example, ten at once; navigate through the strips by using <tt>fs</tt> and <tt>bs</tt>; use <tt>pw</tt> to set the '''assignment y''' in the peak window so that only the assignment in the GFT dimension is displayed. One should see strips from both sub-spectra assigned to the same residue side-by-side.
#In XEASY, use <tt>gs</tt>, <tt>sf</tt>, <tt>fs</tt> and <tt>bs</tt> to go through strips and use <tt>mr</tt> to move the peaks to the actual peak positions. Make sure that the peaks are moved in the right sub-spectrum. If two HB-proton shifts are apparently degenerate, move the corresponding peak to the same position (note that in the better resolved NOESY, the two shifts may turn out to be non-degenerate). If the assignment is ambiguous, load 'central peak spectrum' to identify peak pairs in the sub-spectra. '''Figure 1: Example of (4,3)D GFT HABCAB(CO)NHN analysis before (left) and after (right) peak position adjustment by <tt>mr</tt>.''' [[Image:XEASY hab5.jpg]] [[Image:XEASY hab4.jpg]] 
#In UBNMR, run <tt>updateHabAtom</tt> to update single-quantum 1HAB and 13CAB shifts as <tt>habcabconhO2.prot</tt>. A least-squares fit provides the single-quantum HA/HB chemical shifts (consistency of peaks representing different linear combinations of shifts is checked by UBNMR in oder to identify inadvertently 'mis-picked' peaks; a warning is then provided).
#In XEASY, double-check the peak positions for residues that gaving large error. Repeat previous step and this step till no improvement can be made.
#Now it is ready to move to [[XEASY Side Chain Assignments|Side Chain Assignments]]

 

 

*[[Media:XEASY_makeHabcabPeaks.txt|makeHabcabPeaks]]: UBNMR macro

*[[Media:XEASY_updateHabAtom.txt|updateHabAtom]]: UBNMR macro

HNCACB/CBCA(CO)NH

2009-11-30T21:41:40Z

Jlmills:

=== '''Analysis of the HNNCACB/CACB(CO)NHN''' ===

In case of NMR data were collected with non-GFT NMR experiments such as HNNCACB/CACß(CO)NHN for backbone assignment, one can treat HNNCACB/CACB(CO)NHN the same as previously described [[HNCACAB/CABCA(CO)NH|(4,3)GFT HNNCABCA/CABCA(CO)NHN]] analysis. Simply do the same things as described in analysis of the [[HNCACAB/CABCA(CO)NH|(4,3)D GFT HNNCABCA and CABCA(CO)NHN]] spectra: 

#Simulate the intial peak lists
#Adjust the peak positions in strips using [[XEASY|XEASY]]
#Simulate [[AutoAssign|AutoAssign]] input files and do the initial backbone assignment using [[AutoAssign|AutoAssign]]
#Maually check results from [[AutoAssign|AutoAssign]] and complete the assignment

HNCACAB/CABCA(CO)NH

2009-11-30T21:29:41Z

Jlmills:

== '''Backbone Assignment with XEASY/UBNMR''' ==

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH|HNNCACB/CACBCONHN]].

=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure. 

 

#Creating and adjusting simulated peak lists for (4,3)D GFT spectra 
#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakLists]].
#Edit the [[Media:XEASY_makeCabcaPeak.txt|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]. The new [[XEASY Atom List|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]
##Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> (as described [[XEASY Spin system identification|here]]; you have to use <tt>se</tt> in each spectrum) to sort and display strips (Figure 1A)
##Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
##Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists.

 '''Figure 1: '''Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra '''A: Before peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone1.jpg|1034x766px]] '''B: After peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone2.jpg]]

#'''Initial Backbone Assignment from AutoAssign''' 
##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt|makeAutoList]] and file <tt>myprot.aat</tt>.
##Run <tt>makeAutoList</tt> in [[UBNMR|UBNMR]] to generate input files for [[AutoAssign|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID of the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''
##Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
##Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>. 
##Run [[AutoAssign]] several times (using <tt>Default Execution</tt>) with varying matching tolerances (0.1-0.6ppm) for CA and CB in the <tt>myprot.aat</tt> file and save [[AutoAssign|AutoAssign]] output files 
##From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. 
##In [[UBNMR|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt|AA2Xeasy]] to use the best or consensus [[AutoAssign|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY|XEASY]] SequenceList with <tt>mapping numbers</tt>.
#'''Confirming Backbone Assignment from AutoAssign in XEASY''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that contains only diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; <tt>lc</tt> to load the [[XEASY Atom List|AtomList]]; use <tt>lp</tt> to load the CABCA-[[XEASY Peak List|PeakList]].
##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the [[XEASY Sequence List|SequenceList]] (with [[AutoAssign|AutoAssign]] assignment) and the [[XEASY Atom List|AtomList]]; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues.
##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
##Continue to complete the backbone assignment manually as described below
#'''Completing Backbone Assignment using sequential ordering of SRDs''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
##In XEASY session I, using <tt>fc</tt> and <tt>bc</tt> to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use <tt>sh</tt> to put "on hold".
##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the '''SRD-I''' and '''SRD-II''' numbers to residue numbers.
##In XEASY, repeat 5 and 6 until analysis is complete
##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]] which should be saved, and re-loaded and saved a second time.
##In UBNMR, run [[Media:CleanBbGftProt.txt|cleanBbGftProt]] to make a clean [[XEASY Atom List|AtomList]] and [[XEASY Sequence List|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts. 

 

=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above: 

#Simulating the intial peak lists;
#Adjusting peak position in strips of all residues with XEASY;
#Calculating single quantum chemical shifts from GFT shift linear combinations
#Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
#Maually checking results from AutoAssign and completing the assignment.

File:CleanBbGftProt.txt

2009-11-30T21:27:59Z

Jlmills:

HNCACAB/CABCA(CO)NH

2009-11-30T21:27:17Z

Jlmills:

== '''Backbone Assignment with XEASY/UBNMR''' ==

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH|HNNCACB/CACBCONHN]].

=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure. 

 

#Creating and adjusting simulated peak lists for (4,3)D GFT spectra 
#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakLists]].
#Edit the [[Media:XEASY_makeCabcaPeak.txt|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]. The new [[XEASY Atom List|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]
##Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> (as described [[XEASY Spin system identification|here]]; you have to use <tt>se</tt> in each spectrum) to sort and display strips (Figure 1A)
##Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
##Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists.

 '''Figure 1: '''Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra '''A: Before peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone1.jpg|1034x766px]] '''B: After peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone2.jpg]]

#'''Initial Backbone Assignment from AutoAssign''' 
##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt|makeAutoList]] and file <tt>myprot.aat</tt>.
##Run <tt>makeAutoList</tt> in [[UBNMR|UBNMR]] to generate input files for [[AutoAssign|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID of the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''
##Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
##Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>. 
##Run [[AutoAssign]] several times (using <tt>Default Execution</tt>) with varying matching tolerances (0.1-0.6ppm) for CA and CB in the <tt>myprot.aat</tt> file and save [[AutoAssign|AutoAssign]] output files 
##From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. 
##In [[UBNMR|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt|AA2Xeasy]] to use the best or consensus [[AutoAssign|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY|XEASY]] SequenceList with <tt>mapping numbers</tt>.
#'''Confirming Backbone Assignment from AutoAssign in XEASY''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that contains only diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; <tt>lc</tt> to load the [[XEASY Atom List|AtomList]]; use <tt>lp</tt> to load the CABCA-[[XEASY Peak List|PeakList]].
##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the [[XEASY Sequence List|SequenceList]] (with [[AutoAssign|AutoAssign]] assignment) and the [[XEASY Atom List|AtomList]]; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues.
##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
##Continue to complete the backbone assignment manually as described below
#'''Completing Backbone Assignment using sequential ordering of SRDs''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
##In XEASY session I, using <tt>fc</tt> and <tt>bc</tt> to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use <tt>sh</tt> to put "on hold".
##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the '''SRD-I''' and '''SRD-II''' numbers to residue numbers.
##In XEASY, repeat 5 and 6 until analysis is complete
##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]] which should be saved, and re-loaded and saved a second time.
##In UBNMR, run [[Media:cleanBbGftProt.txt|cleanBbGftProt]]cleanBbGftProt to make a clean [[XEASY Atom List|AtomList]] and [[XEASY Sequence List|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
<blockquote><pre>#UBNMR macro cleanBbGftProt

init

read seq nhsqc.seq

read prot bbgft-swapped.prot append

update atom GFTatom CA

update atom GFTatom CB

remove SRDs

write seq final-clean.seq

remove GFTatoms

write prot final-clean.prot

</pre></blockquote>
 

=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above: 

#Simulating the intial peak lists;
#Adjusting peak position in strips of all residues with XEASY;
#Calculating single quantum chemical shifts from GFT shift linear combinations
#Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
#Maually checking results from AutoAssign and completing the assignment.

File:CleanBBGftProt.txt

2009-11-30T21:24:10Z

Jlmills:

HNCACAB/CABCA(CO)NH

2009-11-30T21:22:45Z

Jlmills:

== '''Backbone Assignment with XEASY/UBNMR''' ==

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH|HNNCACB/CACBCONHN]].

=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure. 

 

#Creating and adjusting simulated peak lists for (4,3)D GFT spectra 
#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakLists]].
#Edit the [[Media:XEASY_makeCabcaPeak.txt|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]. The new [[XEASY Atom List|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]
##Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> (as described [[XEASY Spin system identification|here]]; you have to use <tt>se</tt> in each spectrum) to sort and display strips (Figure 1A)
##Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
##Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists.

 '''Figure 1: '''Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra '''A: Before peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone1.jpg|1034x766px]] '''B: After peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone2.jpg]]

#'''Initial Backbone Assignment from AutoAssign''' 
##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt|makeAutoList]] and file <tt>myprot.aat</tt>.
##Run <tt>makeAutoList</tt> in [[UBNMR|UBNMR]] to generate input files for [[AutoAssign|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID of the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''
##Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
##Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>. 
##Run [[AutoAssign]] several times (using <tt>Default Execution</tt>) with varying matching tolerances (0.1-0.6ppm) for CA and CB in the <tt>myprot.aat</tt> file and save [[AutoAssign|AutoAssign]] output files 
##From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. 
##In [[UBNMR|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt|AA2Xeasy]] to use the best or consensus [[AutoAssign|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY|XEASY]] SequenceList with <tt>mapping numbers</tt>.
#'''Confirming Backbone Assignment from AutoAssign in XEASY''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that contains only diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; <tt>lc</tt> to load the [[XEASY Atom List|AtomList]]; use <tt>lp</tt> to load the CABCA-[[XEASY Peak List|PeakList]].
##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the [[XEASY Sequence List|SequenceList]] that contains [[AutoAssign|AutoAssign]] results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the [[XEASY Sequence List|SequenceList]] (with [[AutoAssign|AutoAssign]] assignment) and the [[XEASY Atom List|AtomList]]; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues.
##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
##Continue to complete the backbone assignment manually as described below
#'''Completing Backbone Assignment using sequential ordering of SRDs''' Here two XEASY sessions are recommended for efficiency: one is for the HNNCABCA/CABCACONHN analysis and the other is for the 15N-resolved NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given '''SRD-I''', one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
##In XEASY session I, using <tt>fc</tt> and <tt>bc</tt> to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use <tt>sh</tt> to put "on hold".
##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the '''SRD-I''' and '''SRD-II''' numbers to residue numbers.
##In XEASY, repeat 5 and 6 until analysis is complete
##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]] which should be saved, and re-loaded and saved a second time.
##In UBNMR, run cleanBbGftProt to make a clean [[XEASY Atom List|AtomList]] and [[XEASY Sequence List|SequenceList]] by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
<blockquote><pre>#UBNMR macro cleanBbGftProt

init

read seq nhsqc.seq

read prot bbgft-swapped.prot append

update atom GFTatom CA

update atom GFTatom CB

remove SRDs

write seq final-clean.seq

remove GFTatoms

write prot final-clean.prot

</pre></blockquote>
 

=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above: 

#Simulating the intial peak lists;
#Adjusting peak position in strips of all residues with XEASY;
#Calculating single quantum chemical shifts from GFT shift linear combinations
#Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
#Maually checking results from AutoAssign and completing the assignment.

HNCACAB/CABCA(CO)NH

2009-11-30T18:06:20Z

Jlmills:

== '''Backbone Assignment with XEASY/UBNMR''' ==

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH|HNNCACB/CACBCONHN]].

=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure. 

 

#Creating and adjusting simulated peak lists for (4,3)D GFT spectra 
#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakLists]].
#Edit the [[Media:XEASY_makeCabcaPeak.txt|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]. The new [[XEASY Atom List|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY Sequence List|SequenceList]], [[XEASY Atom List|AtomList]] and [[XEASY Peak List|PeakList]]
##Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> (as described [[XEASY Spin system identification|here]]; you have to use <tt>se</tt> in each spectrum) to sort and display strips (Figure 1A)
##Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
##Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists.

 '''Figure 1: '''Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra '''A: Before peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone1.jpg|1034x766px]] '''B: After peak position adjustment by <tt>mr</tt>''' [[Image:XEASY backbone2.jpg]]

#'''Initial Backbone Assignment from AutoAssign''' 
##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read [[Media:XEASY_autos_README.txt|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt|makeAutoList]] and file <tt>myprot.aat</tt>.
##Run <tt>makeAutoList</tt> in [[UBNMR|UBNMR]] to generate input files for [[AutoAssign|AutoAssign]]. Errors at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running the <tt>makeAutoList</tt> script again, until all errors are eliminated. The peak ID the simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') corresponds to the '''SRD-I''' number, which is required for this protocol. '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that you generate a 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results. However, please be aware that this protocol discards the 4D information which reduces the GFT (4,3)D spectra into the equivalent of conventional 3D HNNCACB and CBCA(CO)NHN.'''
###Peak pattern used in the [[AutoAssign]] input file for 3D CACB type experiments is: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
###Peak pattern used in the [[AutoAssign]] input file for 4D CACB type experiments is: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt> and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>. 
###Run [[AutoAssign]] several times with varying matching tolerances for CA and CB in the myprot.aat file (0.1-0.6ppm); save [[AutoAssign|AutoAssign]] output files 
###It is suggested to use <tt>Default Execution</tt> method to run the program.
###From the main menu of [[AutoAssign]], select <tt>Examine</tt> > <tt>All GSs</tt> to write an output file that contains both [[AutoAssign|AutoAssign]] assignment and the corresponding '''SRD-I''' residue numbers. 
###In [[UBNMR|UBNMR]], run macro [[Media:XEASY_AA2Xeasy.txt|AA2Xeasy]] to use the best or consensus [[AutoAssign|AutoAssign]] output file to complement the '''SRD-I''' and '''SRD-II''' entries of the [[XEASY|XEASY]] SequenceList with <tt>mapping numbers</tt>.
#'''Confirming Backbone Assignment from AutoAssign in XEASY''' Here two XEASY sessions are recommended for efficiency, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY
##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only contains diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.

##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; <tt>lc</tt> to load the AtomList; use <tt>lp</tt> to load the CABCA-PeakList.
##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the SequenceList (with AutoAssign assignment) and the atomlist; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues in SRD -1 sequential order.
##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
##Continue to complete the backbone assignment manually as described below
#'''Complete Backbone Assignment by perform sequential ordering of SRDs''' Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY.
##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
##In XEASY session I, using <tt>fc</tt> and <tt>bc</tt> to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use <tt>sh</tt> to put "on hold".
##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the SRD-I and SRD-II numbers to residue numbers.
##In XEASY, repeat 5 and 6 until analysis is complete
##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.
##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
<blockquote><pre>#UBNMR macro cleanBbGftProt

init

read seq nhsqc.seq

read prot bbgft-swapped.prot append

update atom GFTatom CA

update atom GFTatom CB

remove SRDs

write seq final-clean.seq

remove GFTatoms

write prot final-clean.prot

</pre></blockquote>
 

=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above: 

#Simulating the intial peak lists;
#Adjusting peak position in strips of all residues with XEASY;
#Calculating single quantum chemical shifts from GFT shift linear combinations
#Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
#Maually checking results from AutoAssign and completing the assignment.

HNCACAB/CABCA(CO)NH

2009-11-30T17:01:40Z

Jlmills:

== '''Backbone Assignment with XEASY/UBNMR''' ==

Sequential backbone and 13CB resonance assignment is associated with mapping of SRDs identified in spin system identification onto the polypeptide sequence. This is accomplished using two (4,3)D GFT NMR experiments, that is, HNNCABCA and CABCA(CO)NHN, or using two non-GFT experiments [[HNCACB/CBCA(CO)NH|HNNCACB/CACBCONHN]].

=== '''Analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra''' ===

The HNNCABCA contains peaks representing both intra-residue and sequential connectivities (as in HNNCACB). Since the sequential connectivities are often comparably weak, this experiment is routinely combined with CABCA(CO)NHN (which comprises, as CBCA(CO)NHN, sequential connectivities only).These can be used to sort SRDs in sequential order, and to then assign them to specific residues in the primary structure. 

#Go to the <tt>analysis/xeasy/backbone</tt> directory and edit the macro <tt>getfil</tt> to import backbone spectra, [[XEASY_Sequence_List|SequenceList]], [[XEASY_Atom_List|AtomList]] and [[XEASY_Peak_List|PeakLists]].
#Edit the [[Media:XEASY_makeCabcaPeak.txt|makeCabcaPeak]] macro and update the sequence and atom list file names. In UBNMR, run the <tt>makeCabcaPeak</tt> script to generate an extended GFT [[XEASY_Atom_List|AtomList]] and [[XEASY_Peak_List|PeakList]]. The new [[XEASY_Atom_List|AtomList]] contains linear combinations of 13CA and 13CB shifts for each residue and SRD. Intraresidue 13C shifts are assigned to '''SRD-I''' numbers, while 13C shifts of the residue preceding '''SRD-I''' are assigned to '''SRD-II''' numbers. This results in a single CABCA-peak list for the four sub-spectra of the two GFT NMR experiments. Peaks are colored according to sub-spectrum and intra- or sequential connectivity. This procedure allows one to efficiently handle sequential connectivities and ensure efficient book-keeping during the assignment process.
#In XEASY, use <tt>ns</tt>, <tt>ls</tt>, <tt>lc</tt>, and <tt>lp</tt> to load HNNCABCA and CABCA(CO)NHN spectra and the [[XEASY_Sequence_List|SequenceList]], [[XEASY_Atom_List|AtomList]] and [[XEASY_Peak_List|PeakList]]
#Use <tt>se</tt>, <tt>gs</tt>, <tt>fs</tt> and <tt>bs</tt> to sort and display strips (Figure 1A)
#Use <tt>mr</tt> to identify and move peaks (Figure 1B); start with CABCA(CO)NHN sub-spectra and continue with HNNCABCA sub-spectra (remove unobserved peaks)
#Use <tt>ra</tt> regularly to check on the quality of the PeakList; <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save updated lists. '''Figure 1: Peak picking of (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra.''' '''A: Before peak position adjustment by <tt>mr</tt>;''' [[Image:XEASY backbone1.jpg]] '''B: After peak position adjustment by <tt>mr</tt>.''' [[Image:XEASY backbone2.jpg]] 

#'''Initial Backbone Assignment from AutoAssign''' 
##Go to <tt>/analysis/xeasy/backbone/autos</tt>, read file [[Media:XEASY_autos_README.txt|autos_README]] for instructions on how to edit the macro [[Media:XEASY_makeAutoList.txt|makeAutoList]] and file <tt>myprot.aat</tt>. In UBNMR run <tt>makeAutoList</tt> to generate input files for AutoAssign. Erros at this step can be corrected by looking closely at the peaks reported, moving them again if needed, and running UBNMR makeAutoList again, until all erros are eliminated. The peak ID of one simulated [[AutoAssign]] input file ('''myprot-hsqc.pks''') correspond to the residue number of '''SRD-I''', which is required for this protocol. '''Since HNNCABCA and CABCA(CO)NHN spectra provide 4D information, it is suggested that generate the 4D peak list for AutoAssign in order to take full advantage of GFT spectra and get a better assignment results.'''
##:*Click for the [[AutoAssign]] control file example [[Media:XEASY_myprot.aat|myprot.aat]].
##:*Peak pattern used in the [[AutoAssign]] input file of 3D CACB type experiments: <tt>HN(i), N(i), CA(i or i-1) or CB(i or i-1)</tt>;
##:*Peak pattern used in the [[AutoAssign]] input file of 4D CACB type experiments: <tt>HN(i), N(i), CA(i), CA(i) or CB(i)</tt>; and <tt>HN(i), N(i), CA(i-1), CA(i-1)</tt> or <tt>CB(i-1)</tt>. 
##Run [[AutoAssign]] several times with varying matching tolerances for CA and CB in the myprot.aat file (0.1-0.6ppm); save AutoAssign output files; 
##:*It is suggested to use <tt>Default Execution</tt> method to run the program.
##:*From the main menu of [[AutoAssign]], follow option <tt>Examine</tt> > <tt>All GSs</tt> to write output file that contains both AutoAssign assignment and the corresponding '''SRD-I''' residue numbers. 
##In UBNMR, run macro [[Media:XEASY_AA2Xeasy.txt|AA2Xeasy]] to use the best or consensus AutoAssign output file to complement the SRD-I and SRD-II entries of the XEASY SequenceList with <tt>mapping numbers</tt>. 

 Modified macro for AA2XEASY
<blockquote><pre>#UBNMR script for converting autoassign results to xeasy format sequence

init read seq ../nhsqc.seq

#update autoassign results to xeasy sequence

update mapping aa.out 200

#200 is the number difference between self and sequencial SRD's.

write updated sequence

write sequence aa.seq
</pre></blockquote>
 

#'''Confirming Backbone Assignment from AutoAssign in XEASY''' Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY '''Also see below the use Of 15N-resolved [1H,1H] NOESY Spectrum).''' 
##In UBNMR, run macro [[Media:XEASY_makeBbNNoesy.txt|makeBbNNoesy]] to use the 15N /1HN backbone shifts of SRD-I to generate a starting peaklist that only contains diagonal peaks for analysis of the 15N-resolved part of simultaneous 3D 15N/13Caliphatic/13Caromatic-resolved [1H,1H]-NOESY.
##In XEASY session I, <tt>ns</tt> to load the four sub-spectra of (4,3)D HNNCABCA/CABCACONHN; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; <tt>lc</tt> to load the AtomList; use <tt>lp</tt> to load the CABCA-PeakList.
##In XEASY session II, use <tt>ns</tt> to load 15N-resolved NOESY; use <tt>ls</tt> to load the SequenceList that contains AutoAssign results; use <tt>lp</tt> to load the corresponding starting peak list. There is no need to move any peaks at this time.
##In XEASY session I and II, use <tt>sn</tt> to swap fragment number and mapping; use <tt>se</tt> to sort strips; use <tt>ls</tt> and <tt>lc</tt> to load the SequenceList (with AutoAssign assignment) and the atomlist; use <tt>gs</tt> to display the strips. Now the strips are sorted in the following order: Assigned SRD residues in sequential order followed by unassigned SRD residues in SRD -1 sequential order.
##In XEASY session I and II, check and confirm sequential connectivities for the assigned SRD residues. Use <tt>ed</tt> to modify the mapping number if the assignment is wrong.
##Continue to complete the backbone assignment manually as described below 
#'''Complete Backbone Assignment by perform sequential ordering of SRDs''' Here two XEASY sessions are recommended for efficiency reasons, one is HNNCABCA/CABCACONHN analysis, the other is 15N NOESY analysis. For each [w1(13CA;13CAB),w3(1HN)]-strip corresponding to the 15N/1HN shifts of a given SRD-1, one expects to observe up to four [two] peaks in the two sub-spectra of (4,3)D HNNCABCA[CABCA(CO)NHN]. In the following, sequential ordering of these strips in XEASY is described. This leads to "sequential walks" in the two sub-spectra along the polypeptide chain. Sequential connectivities are confirmed in 15N-resolved NOESY.
##In XEASY session I, use <tt>es</tt>, <tt>se</tt>, <tt>gs</tt> to select two strips exhibiting four well resolved peaks arising from a Ser, Thr, Ala residue in (4,3)D HNNCABCA or terminal residues of an assigned segment assigned from AutoAssign as a starting point; use <tt>sh</tt> to put those strips "on hold".
##In XEASY session I, use <tt>rd</tt> and <tt>pc</tt> to search for sequential neigbours.
##In XEASY session I, using <tt>fc</tt> and <tt>bc</tt> to inspect the sorted strips in order to identify the strip containing the sequential neighbor and use <tt>sh</tt> to put "on hold".
##In XEASY session II, use <tt>cd</tt> / <tt>cc</tt> to confirm sequential connectivities in 15N-resolved NOESY.
##In XEASY, repeat steps 1 to 4 until you have identified a maximal set of strips you can map to the polypeptide sequence.
##In XEASY, use <tt>ed</tt> to edit peak entries, type in "mapping numbers" which link the SRD-I and SRD-II numbers to residue numbers.
##In XEASY, repeat 5 and 6 until analysis is complete
##In XEASY, use <tt>aa</tt>, <tt>ac</tt>, <tt>ws</tt>, <tt>wc</tt> and <tt>wp</tt> to save all XEASY files before switching from SRD to sequential amino acid residue numbering using <tt>sn</tt>. This yields modified SequenceList, AtomList and PeakList which should be saved, and re-loaded and saved a second time.
##In UBNMR, run <tt>cleanBbGftProt</tt> to make a clean AtomList and SeqList by deleting extra atoms and SRDs, fixing nomenclature, and updating single-quantum 13CA and 13CA shifts.
<blockquote><pre>#UBNMR macro cleanBbGftProt

init

read seq nhsqc.seq

read prot bbgft-swapped.prot append

update atom GFTatom CA

update atom GFTatom CB

remove SRDs

write seq final-clean.seq

remove GFTatoms

write prot final-clean.prot

</pre></blockquote>
 

=== '''Analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra''' ===

In case of NMR data were collected with (4,2)D GFT HNNCABCA and CABCA(CO)NHN for backbone assignment, one can treat them the same as previously described (4,3)D GFT HNNCABCA/CABCA(CO)NHN analysis. Since the protocol of analysis of the (4,3)D GFT HNNCABCA and CABCA(CO)NHN spectra is based on analysis the stips residue by residue, it can be completely adapted to the analysis of the(4,2)D GFTHNNCABCA and CABCA(CO)NHN Spectra. Simply do the same things as described above: 

#Simulating the intial peak lists;
#Adjusting peak position in strips of all residues with XEASY;
#Calculating single quantum chemical shifts from GFT shift linear combinations
#Simulating AutoAssign input files and do the initially backbone assignment by AutoAssign;
#Maually checking results from AutoAssign and completing the assignment.

Wiki Tree Layout

2009-11-24T20:19:35Z

Jlmills:

This outline of the NESG NMR Wiki is designed to expand on the existing "Master Recipe" and should serve as an experience harvesting tool.

*It has a rather broad coverage to facilitate long-tewrm growth and development. Aditional compact aggregator pages may be needed to pesent specific information concisely.
*There would be separate webs within the wiki: Public(or Main), NESG, and member lab webs. Most common knowlege topics should be public, unless they are specific to NESG
*We assume that the target audience has some knowledge about NMR and protein structure determination, but make the content useful for training
*"Resonance Assignment" and "Structure Determination" chaptes would focus on individual software packages. The XEASY resonance assignment tree, as the most complete, would serve as a template for other software.
*Most chapters should include a "general principles" section.

Please leave your comments/suggestion at the bottom of this page

 

= HTP NMR structure determination =

== Protein Target Selection, Sample Preparation, and Initial Screening ==

#[[Target selection|NESG target selection]] - overview of target selection in PSI III
#[[Bioinformatics with protein sequence]]
#[[DNA cloning protocols|DNA cloning protocols]] 
#[[Protein purification|Protein expression and purification protocols]]  
#Sample Optimization
##[[Construct optimization]]
##[[Buffer optimization]]
##[[Cofactor optimization]]
#Initial protein analysis
##[[SDS page gel]]
##[[Protein concentration|Protein concentration measurements]]
##[[Oligomerization Status|Assessment of Oligomerization Status]]
###[[Gel filtration and light scattering|gel-filtration and light scattering]]
###[[Sedimentation equilibrium|Sedimentation equilibrium]]
###[[NMR determined Rotational correlation time]]
##[[MassSpectrometry|Mass spectrum]]
##NMR screening
###[[1D screening|1H 1D screening]]
###[[Nhsqc screen|Initial [15N,1H] HSQC]] 

== NMR Data Collection ==

#Routine operation
##[[NMR sample tubes]]
##[[NMR Sample Preparation]]
##[[Inserting NMR Sample]]
##Tuning and matching
##[[Deuterium Lock]]
##[[Shimming]]
##[[Pulse width calibration]]
##[[Temperature calibration]]
##[[Chemical shift referencing]]
#Advanced operation
##[[Deuterium pulse width calibration and decoupling]]
#NMR data acquisition for protein structure determination
##[[Common NMR experiment sets]]
##Custom NMR experiment setup scripts for VNMRJ
##1D 1H NMR spectra and 2D [15N, 1H]-HSQC
##[[Estimation of rotational correlation time]]
##Estimation of measurement time
##NMR experiments for spin system identification
##2D and 3D NOESY
##Double and triple NMR experiments
###3D CBCA(CO)NH and HNCACB
###3D HNCA and HN(CO)CA
###3D HAHB(CO)NH
###(4,3)D CABCA(CO)NH and HNCACB
###(4,3)D HABCAB(CO)NH
###(H)CCH
###(H)CCH-TOCSY
###H(C)CH
###H(C)CH-TOCSY
###(4,3)D HCCH
##Other NMR experiments
###2D [13C, 1H]-HSQC for 5% 13C-labeled samples
###2D [15N, 1H]-long-range-HSQC for determination of histidine protomer state
###MEXICO
###CLEANEX
###H-D exchange experiment
###15N spin relaxation parameters
#Advanced problems for data collection
##[[Setting up non-uniformly sampled spectra]]
###[[NUS guide for Varian|Guide for Varian/BioPack]]
###[[NUS guide for Bruker according to Arrowsmith group in Toronto|Guide for Bruker/Topspin according to Arrowsmith group]]
#Maintenance
##VARIAN
###Installing and updating BioPack
###Full Probefile calibration
###Rebooting the console
###Cryoprobe conditioning
##BRUKER

== NMR Data Processing ==

#General Priciples and Concepts
##Fourier transformation
###Zero-filling
###Apodization
###Phasing
###Linear prediction
###G-matrix Fourier transformation (GFT)
##Alternatives to Fourier transformation
###Maximum entropy reconstruction
###[[Processing non-uniformly sampled spectra with Multidimensional Decomposition]]
###...
#Practical Aspects
##NMRPIPE
###General information
###Buffalo's Processing Protocol using NMRpipe
##PROSA
##TOPSPIN
##[[AGNuS/AutoProc|AGNuS/AutoProc]]
##UBNMR
##Spectral format conversion

 

== Resonance Assignment ==

This chapter would focus on individual data analysis and resonance assignment packages, as most people stick to a particular software for entire structure determination projects.

 

#[[Principles and concepts]]
##[[Stable isotope labeling schemes]]
##[[NMR experiments]]
###[[Through-bond]]
###[[Through space]]
##[[Spin systems]]
###[[Definitions]]
###[[Identification]]
###[[Linking spin systems]]
###[[Matching onto covalent structure]]
#[[Practical aspects]]
##[[Semi-automated protocols]]
###[[CARA]]
####[[Spin System Identification with CARA|Spin System Identification in 2D 15N-HSQC and 3D HNNCO]]
####[[Backbone Assignment with CARA|Backbone Resonance Assignment]]
####[[HA and HB Assignment with CARA|Assignment of HA and HB Resonances with (4,3)D GFT HABCAB(CO)NHN]]
####Side Chain Assignment
#####[[Aliphatic Side Chain Assignment with CARA|Aliphatic side-chain assignment]]
#####[[Aromatic Side Chain Assignment with CARA|Aromatic side-chain assignment]]
#####[[Amide Side Chain Assignment with CARA|Amide side-chain assignment]]
###[[Sparky]]
###[[XEASY]]
####[[XEASY Spin system identification|Spin system identification]]
####Backbone resonance assignment''' '''
#####GFT-based spectra
######[[HNCACAB/CABCA(CO)NH]]
#####Conventional spectra
######[[HNCACB/CBCA(CO)NH]]
######HNCA/HN(CO)CA
######HNCO/HN(CA)CO
######NOESY/TOCSY
####[[XEASY Side Chain Assignment|Side chain resonance assignment]]
#####Aliphatic
######GFT NMR spectra
#######[[HA and HB Assignment with GFT in XEASY|(4,3)D GFT HABCAB(CO)NHN]]
#######[[Side chain assignment with aliphatic (4,3)D HCCH-COSY in XEASY|(4,3)D GFT HCCH]]
######Conventional spectra
#######HAHB(CO)NH
#######HCCH-COSY
#######HCCH-TOCSY
#######[[Side chain assignment with CN-NOESY in XEASY|Simultaneous NOESY]]
#######(H)CC-TOCSY-(CO)NH
#######H(CC-TOCSY-CO)NH
#####[[Aromatic side chain assignment with Aro-HCCH-COSY in XEASY|Aromatic]]
######GFT-based spectra
######Conventional spectra
#####Other
######Trp e1 NH and d1 CH
######[[Met methyl assignment with NOESY|Met e CH3 ]]
######[[Amide Side Chain assignment with NOESY|Asn d2 and Gln e2 NH2]]
#####NOESY peak integration
##Automated protocols
###[[AutoAssign|AutoAssign]]
###[[AutoAssign WebServer|AutoAssign server]]
###[[Abacus|ABACUS]]
###[[The PINE Server|PINE server]]
##Validation of resonance assignment
###[[AVS|Assignment validation suite (AVS)]]
###[[LACS|Linear analysis of chemical shift (LACS)]]
##Depositing chemical shifts

 

== Structure Calculation and Validation ==

#[[Structure Calculation and Validation|Principles and concepts]]
#Practical aspects
##Structure calculation
###CYANA
####[[CYANA|Getting started]]
####[[FOUND|FOUND]]
####[[TALOS|TALOS]]
####[[GLOMSA|GLOMSA]]
####[[NOE Calibration Using CYANA|NOE calibration]]
####[[Manual Structure Calculation Using CYANA|Manual structure calculation]]
####[[Automated NOESY Assignment Using CYANA|Automated NOESY assignment and structure calculation]]
####[[Structure Calculation With RDC's Using CYANA|Structure calculation with residual dipolar couplings]] (link to REDCAT/PALES,FINDTENSOR, .rdc file, adding ORI to PDB file)
####[[Homodimer Structure Calculation Using CYANA|Homodimer structure calculations]][[Homodimer Structure Calculation Using CYANA| ]]
###AutoStructure
####[[AutoStructure/RPF Theory|Theory]]
####[[AutoStructure|Getting started]]
####[[CYANA Structure Calculations Using AutoStructure|CYANA run]]
####[[XPLOR Structure Calculations Using AutoStructure|XPLOR run]]
####[[Analyzing AutoStructure Output Directories|Analyzing the output]]
####[[RPF Analysis|RPF/DP scores]]
####[[Structure Calculation Using AS-DP|Structure calculation using AS-DP]]
###"Consensus" Approaches
####[[Overview of Consensus Runs|Overview of Consensus runs]]
####[[Finding Consensus NOE Assignments|Finding Consensus NOE assignments]]
####[[Validation of Consensus Run|Validation of Consensus runs]]
###[[Structure Calculation Using CS-Rosetta|CS-ROSETTA]]
###[[Structure Calculation Using CS-CP ROSETTA|CS-DP ROSETTA]]
###[[Structure Calculation Using RDC-ROSETTA|RDC-ROSETTA]]
###[[RDC-Assisted Dimer Structure Determination|RDC-assisted dimer structure calculation]] 
###Special topics
####[[Protein-Ligand Complexes|Protein-Ligand complexes]]
####[[Working With Metal Ions|Metal ions]]
####[[Working With Dimers|Dimers]]
####[[Residual Dipolar Couplings in Structure Refinement|Residual Dipolar Couplings]]
####[[REDCAT|REDCAT]] and [[REDCRAFT|REDCRAFT]]
####[[Paramagnetic Constraints in Structure Determination|Paramagnetic constraints]]
##Structure Refinement
###[[Structure Refinement Using CNS Energy Minimization With Explicit Water|CNS refinement]]
###[[Structure Refinement Using XPLOR-NIH|XPLOR-NIH refinement]]
###[[Rosetta High Resolution Protein Structure Refinement Protocol|ROSETTA refinement]]
##Validation and deposition
###[[PdbStat|PdbStat]]
###[[PSVS|PSVS]]
###[[RPF Analysis|RPF analysis]]
###[[MolProbity Server|MolProbity server]]
###[[PDB and BMRB Deposition|PDB and BMRB deposition]]
###[[ADIT-NMR|ADIT-NMR]]
###[[HarvestDB|HarvestDB]]
###[[SPINS|SPINS]]

-- JeffMills - 28 May 2009

Here are two comments from Guy:

- need to have centralized site for downloading all software that NESG has developed or licensed; this would be a central site for NESG scientists to use to access the latest version of all software

- need to allow outside users to access links to all software (they will need licenses to download) and also to download software from NESG

-- AlexEletski - 13 Jul 2009

XEASY Backbone Assignment

2009-11-24T20:18:54Z

Jlmills: Blanked the page

XEASY Spin system identification

2009-11-24T18:33:26Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY Sequence List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY|XEASY]] Files for book-keeping: [[XEASY Atom List|AtomList]], [[XEASY Sequence List|SequenceList]], and [[XEASY Peak List|PeakList]].
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY|XEASY]] library and [[XEASY Sequence List|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY Atom List|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY Peak List|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY Atom List|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY Atom List|AtomList]] entries corresponding to '''SRD-I'''.

 '''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

'''A: After in-phase peak picking''' [[Image:XEASY hsqc2.jpg]] 

'''B: XEASY <tt>ar</tt> window''' [[Image:XEASY hsqc ar.jpg]] 

 '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>''' 

[[Image:XEASY hsqc3.jpg]] 

 

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
#Run the <tt>makeHncoPeak</tt> script (see below) in [[UBNMR|UBNMR]].This [[XEASY Peak List|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY Peak List|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
#Start [[XEASY|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY Sequence List|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY Atom List|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY Peak List|PeakList]] (<tt>hncoI1.peak</tt>). 
#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (a<tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY Atom List|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY Peak List|PeakList]] as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY Backbone Assignment|backbone assignment]].

 

An example of the <tt>makeHncoPeak</tt> [[UBNMR|UBNMR]] script
<pre>init
read seq nhsqcO1.seq
read prot nhsqcO1.prot
write prot hncoI1.prot
simulate 3D N C -1 2
write peaks hncoI1.peaks
</pre>
 

 '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip''' [[Image:XEASY hnco1.jpg]]

 '''B: After <tt>mr</tt>''' [[Image:XEASY hnco2.jpg]] 
====

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory. 
#Run <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.
#Follow the protocol shown above for assignment of 3D HNNCO spectra. 

 

An example of the <tt>make32DHncoPeak</tt> [[UBNMR|UBNMR]] script
<pre>init
read seq nhsqcO1.seq
read prot nhsqcO1.prot
write prot hncoI1.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks hncoI1.peaks
</pre>
 -- Main.GaohuaLiu - 16 Feb 2007

XEASY Spin system identification

2009-11-24T18:31:06Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY Sequence List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY|XEASY]] Files for book-keeping: [[XEASY_Atom_List|AtomList]], [[XEASY_Sequence_List|SequenceList]], and [[XEASY_Peak_List|PeakList]].
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY|XEASY]] library and [[XEASY_Sequence_List|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY_Atom_List|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY_Peak_List|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY_Atom_List|AtomList]] entries corresponding to '''SRD-I'''.

 '''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

'''A: After in-phase peak picking''' [[Image:XEASY hsqc2.jpg]] 
 

'''B: XEASY <tt>ar</tt> window''' [[Image:XEASY hsqc ar.jpg]] 

 '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>''' 

[[Image:XEASY hsqc3.jpg]] 

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
#Run the <tt>makeHncoPeak</tt> script (see below) in [[UBNMR|UBNMR]].This [[XEASY Peak List|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY Peak List|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
#Start [[XEASY|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY Sequence List|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY Atom List|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY Peak List|PeakList]] (<tt>hncoI1.peak</tt>). 
#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (a<tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY Atom List|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY Peak List|PeakList]] as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY Backbone Assignment|backbone assignment]]

An example of the <tt>makeHncoPeak</tt> [[UBNMR|UBNMR]] script
<pre>init
read seq nhsqcO1.seq
read prot nhsqcO1.prot
write prot hncoI1.prot
simulate 3D N C -1 2
write peaks hncoI1.peaks
</pre>
 

 '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip''' [[Image:XEASY hnco1.jpg]]

 '''B: After <tt>mr</tt>''' [[Image:XEASY hnco2.jpg]] 

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory 
#Run <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.
#Follow the protocol shown above for assignment of 3D HNNCO spectra. 

An example of the <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] script
<pre>init
read seq nhsqcO1.seq
read prot nhsqcO1.prot
write prot hncoI1.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks hncoI1.peaks
</pre>

-- Main.GaohuaLiu - 16 Feb 2007

XEASY Spin system identification

2009-11-24T18:29:21Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY Sequence List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY|XEASY]] Files for book-keeping: [[XEASY_Atom_List|AtomList]], [[XEASY_Sequence_List|SequenceList]], and [[XEASY_Peak_List|PeakList]].
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY|XEASY]] library and [[XEASY_Sequence_List|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY_Atom_List|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY_Peak_List|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY_Atom_List|AtomList]] entries corresponding to '''SRD-I'''.

 '''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

'''A: After in-phase peak picking''' [[Image:XEASY hsqc2.jpg]] 
 

'''B: XEASY <tt>ar</tt> window''' [[Image:XEASY hsqc ar.jpg]] 

 '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>''' 

[[Image:XEASY hsqc3.jpg]] 

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
#Run the <tt>makeHncoPeak</tt> script in [[UBNMR|UBNMR]].This [[XEASY_Peak_List|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY_Peak_List|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
#Start [[XEASY|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY_Sequence_List|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY_Peak_List|PeakList]] (<tt>hncoI1.peak</tt>). 
#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (a<tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY_Atom_List|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY_Peak_List|PeakList]] as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY_Backbone_Assignment|backbone assignment]]

 '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip''' [[Image:XEASY hnco1.jpg]]

 '''B: After <tt>mr</tt>''' [[Image:XEASY hnco2.jpg]] 

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

#Go to the <tt>/protName/analysis/xeasy/hnco</tt> directory 
#Run <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] (see below) to create a (3,2)D GFT HNNCO peaklist.
#Follow the protocol shown above for assignment of 3D HNNCO spectra. 

An example of the <tt>make32DHncoPeak</tt> in [[UBNMR|UBNMR]] script
<pre>init
read seq nhsqcO1.seq
read prot nhsqcO1.prot
write prot hncoI1.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks hncoI1.peaks
</pre>

-- Main.GaohuaLiu - 16 Feb 2007

XEASY Spin system identification

2009-11-24T18:23:54Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY Sequence List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY|XEASY]] Files for book-keeping: [[XEASY_Atom_List|AtomList]], [[XEASY_Sequence_List|SequenceList]], and [[XEASY_Peak_List|PeakList]].
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY|XEASY]] library and [[XEASY_Sequence_List|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY_Atom_List|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY_Peak_List|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY_Atom_List|AtomList]] entries corresponding to '''SRD-I'''.

 '''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

'''A: After in-phase peak picking''' [[Image:XEASY hsqc2.jpg]] 
 

'''B: XEASY <tt>ar</tt> window''' [[Image:XEASY hsqc ar.jpg]] 

 '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>''' 

[[Image:XEASY hsqc3.jpg]] 

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/hnco</tt> and make a copy of HNCO spectrum (<tt>cp HNCO1.3D.16 </tt><tt>HNCO1a.3D.16</tt> and <tt>cp HNCO1.3D.param </tt><tt>HNCO1a.3D.param</tt>).
#Run the <tt>makeHncoPeak</tt> script in [[UBNMR|UBNMR]].This [[XEASY_Peak_List|PeakList]] will contain one peak for each amide N(i) / H(i) moiety which will be assigned to an '''SRD-I''' number derived from the 2D [15N,1H]-HSQC [[XEASY_Peak_List|PeakList]]. The third / 13C'(i-1) dimension is assigned to the corresponding '''SRD-II''' number and has a default chemical shift of 175ppm.
#Start [[XEASY|XEASY]] and use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutations set to <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. Use <tt>ls</tt> to load the [[XEASY_Sequence_List|SeqList]] (<tt>nhsqc.seq</tt>), <tt>lc</tt> to load the [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>), <tt>lp</tt> to load the [[XEASY_Peak_List|PeakList]] (<tt>hncoI1.peak</tt>). 
#Use <tt>cp</tt> to display the spectrum as a contour plot. Next, create the strips using <tt>se</tt> in the <tt>HNCO1</tt> spectrum, then change to <tt>HNCO1a</tt> using <tt>rc</tt> and <tt>rs</tt>, and create strips using <tt>se</tt> again in the <tt>HNCO1 </tt>spectrum. Use <tt>gs</tt> to display the strips (Figure 2A).
#Use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B).
#Use <tt>dp</tt> to delete predicted peak markers that do not correspond to actual peaks in the spectrum (side-chain peaks) and <tt>pp</tt> to manually pick and assign (a<tt>p</tt>) newly observed peaks (usually a result of overlap in the 2D [15N, 1H]-HSQC).
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the [[XEASY_Atom_List|AtomList]] as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the [[XEASY_Peak_List|PeakList]] as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY_Backbone_Assignment|backbone assignment]]

 '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip''' [[Image:XEASY hnco1.jpg]]

 '''B: After <tt>mr</tt>''' [[Image:XEASY hnco2.jpg]] 

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

*Go to <tt>/protName/analysis/xeasy/hnco</tt>
*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist. 
<blockquote><pre>init
read seq nhsqc.seq
read prot noe.prot

write prot hnco.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks HNNCO.peaks
</pre></blockquote>
*Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

 -- Main.GaohuaLiu - 16 Feb 2007</pre>

XEASY Spin system identification

2009-11-24T18:01:20Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[Fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY Sequence List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY Sequence List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See [[XEASY|XEASY]] Files for book-keeping: [[XEASY_Atom_List|AtomList]], [[XEASY_Sequence_List|SequenceList]], and [[XEASY_Peak_List|PeakList]].
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, [[XEASY|XEASY]] library and [[XEASY_Sequence_List|SequenceList]]. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial [[XEASY_Atom_List|AtomList]] is generated automatically. Use <tt>in</tt> for automatic in-phase peak picking of the 2D [15N,1H]-HSQC spectrum. Complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to pick additional peaks (Figure.1A). Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C). Occasionally, the <tt>ar</tt> command causes an error <tt>Atom N 201 not known!</tt> which can be solved by loading the library file [<tt>ll</tt>] from <tt>/nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib</tt> and then repeating the <tt>ar</tt> command. Average the chemical shifts and save the [[XEASY_Peak_List|PeakList]] (<tt>nhsqcO1.peaks</tt>) and [[XEASY_Atom_List|AtomList]] (<tt>nhsqcO1.prot</tt>) using <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt>. Then, amide 15N / 1HN chemical shifts are transferred into the [[XEASY_Atom_List|AtomList]] entries corresponding to '''SRD-I'''.

 '''Figure 1.''' Peak picking the 2D [15N, 1H]-HSQC spectrum

'''A: After in-phase peak picking''' [[Image:XEASY hsqc2.jpg]] 
 

'''B: XEASY <tt>ar</tt> window''' [[Image:XEASY hsqc ar.jpg]] 

 '''C: After assigning the peaks to SRD-I numbers using <tt>ar</tt>''' 

[[Image:XEASY hsqc3.jpg]] 

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Run the <tt>makeHncoPeak</tt> script in [[UBNMR|UBNMR]]. A starting peak list for analysis of (3,2)D HNNCO or 3D HNNCO is generated as <tt>hncoI1.peaks</tt>.
#In XEASY, perform HNNCO Analysis (described in HNNCO analysis).
#Go to <tt>/protName/analysis/xeasy/hnco</tt>
#In the spectra folder, make a identical copy of HNCO spectrum, eg. cp spectrum <tt>HNCO1</tt> to spectrum <tt>HNCO1a</tt>.
#In UBNMR, run <tt>makeHncoPeak</tt> to produce the file <tt>hncoI1.peaks</tt>. This <nop>PeakList will contain one peak for each amide N,H moiety assigned to SRD-I derived from the 2D [15N,1H]-HSQC <nop>PeakList. 175 ppm is assigned to all 13C' shifts of SRD-II.
#In XEASY, use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutation <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. from <tt>/protName/xeasy/data</tt>; use <tt>ls</tt> to load the sequence file, <tt>nhsqc.seq</tt>; use <tt>lc</tt> to load the atom list (protlist), <tt>nhsqcO1.prot</tt>; use <tt>lp</tt> to load the initial HNCO peaklist from <tt>hncoI1.peak</tt>; use <tt>cp</tt> to display the spectrum as a contour plot; use <tt>se</tt> to create strips for all of the peaks in the peaklist; use <tt>gs</tt> to display the first set of strips (Figure 2A). '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip;''' [[Image:XEASY hnco1.jpg]] 
#In XEASY, use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B); use <tt>dp</tt> to delete unobserved predicted peaks (side-chain peaks), and use <tt>pp</tt> manually pick and assign observed unpredicted peaks (overlapped in Nhsqc). '''B: After <tt>mr</tt>;''' [[Image:XEASY hnco2.jpg]] 
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the atom list as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the peaklist as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY Backbone Assignment|backbone assignment]]

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

*Go to <tt>/protName/analysis/xeasy/hnco</tt>
*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist. 
<blockquote><pre>init
read seq nhsqc.seq
read prot noe.prot

write prot hnco.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks HNNCO.peaks
</pre></blockquote>
*Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

 -- Main.GaohuaLiu - 16 Feb 2007</pre>

XEASY Spin system identification

2009-11-24T16:53:24Z

Jlmills:

== Spin System Identification ==

2D [15N,1H]-HSQC provide pairs of correlated amide 15N / 1HN chemical shifts. They seed '''spin systems''' - spins of individual residues, whose assignment to the protein sequence is normally not known ''a priori''. In an [[XEASY|'''XEASY''']]-based approach, spin systems are called '''SRD''' spin systems.

(3,2)D HNNCO or 3D HNNCO provide additional resolution when both 15N and 1HN chemical shifts overlap, and help exclude side-chain peaks. Although 13C' chemical shifts are seldom used for sequence-specific assignment, they are used by the programs [[CSI|'''CSI''']] and [[TALOS|'''TALOS''']].

=== '''Spin System Identification with XEASY/UBNMR''' ===

==== '''Peak Picking the (15N,1H) HSQC Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/nhsqc.</tt> Create a file with the amino acid sequence in [[fasta|FASTA]] format, obtained, for example, from the [http://spine.nesg.org/ SPINE] web page, and save it as <tt>aa.seq</tt>. Run the <tt>makeSeq</tt> macro with [[UBNMR|UBNMR]] to generate an [[XEASY|XEASY]] [[XEASY_Sequence_List|SequenceList]] as <tt>nhsqc.seq</tt> . This [[XEASY_Sequence_List|SequenceList]] contains entries for the amino acids residues first, followed by two sets of '''SRD'''s named in the following way: '''SRD-I''' (Starting from 201) and '''SRD-II''' (starting from 401). '''SRD-II''' entires serve to handle sequential connectivities. See XEASY Files for Book-keeping: <nop>AtomList, <nop>SequenceList, <nop>PeakList.
#In XEASY, use <tt>ns</tt>, <tt>ll</tt> and <tt>ls</tt> to load 2D [15N,1H]-HSQC spectrum, XEASY library and <nop>SequenceList. To change display from the default one to contour plot, type <tt>cp</tt> and give the appropriate threshold level. A corresponding initial <nop>AtomList is generated automatically; use <tt>in</tt> for in-phase 'peak picking' of the 2D [15N,1H]-HSQC spectrum automatically; complete peak picking manually by using <tt>dp</tt> to remove the peaks belonging to sidechain amides which can be identified by a NH2only HSQC if the region is crowded and <tt>pp</tt> to 'peak pick' additional peaks (Figure.1A); Use <tt>ar</tt> to automatically assign each peak to the backbone amide moiety of '''SRD-I''' residues (Figure.1B,C); use <tt>ac</tt>, <tt>wc</tt> and <tt>wp</tt> to save the updated <nop>AtomList as <tt>nhsqcO1.prot</tt> and <nop>PeakList as <tt>nhsqcO1.peaks</tt>. Then, 15N / amide 1HN chemical shifts are transferred into the <nop>AtomList entries corresponding to '''SRD-I'''. At times the <tt>ar</tt> command gives an error "Atom N 201 not known!". This can be rectified by loading a different library file [ll] from /nsm/chem/cen2/HTP2/3_src/xeasy/src.new/xeasy.lib and then trying to auto assign the peaks. '''Figure 1.''' Peak picking the NHSQC spectrum*

'''A: After in-phase peak picking; ''' [[Image:XEASY hsqc2.jpg]] 

'''B: XEASY <tt>ar</tt> window'''

 [[Image:XEASY hsqc ar.jpg]] 

 '''C: After XEASY command <tt>ar</tt> .'''

 

[[Image:XEASY hsqc3.jpg]] 

#In UBNMR run <tt>makeHncoPeak</tt>. A starting peak list for analysis of (3,2)D HNNCO or 3D HNNCO is generated as <tt>hncoI1.peaks</tt>. /
#In XEASY, perform HNNCO Analysis (described in HNNCO analysis).

==== '''Analysis of the 3D HNNCO Spectrum''' ====

#Go to <tt>/protName/analysis/xeasy/hnco</tt>
#In the spectra folder, make a identical copy of HNCO spectrum, eg. cp spectrum <tt>HNCO1</tt> to spectrum <tt>HNCO1a</tt>.
#In UBNMR, run <tt>makeHncoPeak</tt> to produce the file <tt>hncoI1.peaks</tt>. This <nop>PeakList will contain one peak for each amide N,H moiety assigned to SRD-I derived from the 2D [15N,1H]-HSQC <nop>PeakList. 175 ppm is assigned to all 13C' shifts of SRD-II.
#In XEASY, use <tt>ns</tt> to load both HNNCO spectra <tt>HNCO1</tt> and <tt>HNCO1a</tt> with permutation <tt>x:HN, y:C, Z:N</tt> and <tt>x:N, y:C, Z:HN</tt>, respectively. from <tt>/protName/xeasy/data</tt>; use <tt>ls</tt> to load the sequence file, <tt>nhsqc.seq</tt>; use <tt>lc</tt> to load the atom list (protlist), <tt>nhsqcO1.prot</tt>; use <tt>lp</tt> to load the initial HNCO peaklist from <tt>hncoI1.peak</tt>; use <tt>cp</tt> to display the spectrum as a contour plot; use <tt>se</tt> to create strips for all of the peaks in the peaklist; use <tt>gs</tt> to display the first set of strips (Figure 2A). '''Figure 2: Analysis of the 3D HNNCO by XEASY''' '''A: Before <tt>mr</tt>, showing orthogonal views of each strip;''' [[Image:XEASY hnco1.jpg]] 
#In XEASY, use <tt>mr</tt> to move each pre-positioned peak onto the actual peak (Figure 2B); use <tt>dp</tt> to delete unobserved predicted peaks (side-chain peaks), and use <tt>pp</tt> manually pick and assign observed unpredicted peaks (overlapped in Nhsqc). '''B: After <tt>mr</tt>;''' [[Image:XEASY hnco2.jpg]] 
#After all peaks are 'picked', use <tt>ac</tt> to update the chemical shifts, <tt>wc</tt> to save the atom list as <tt>hncoO1.prot</tt>, and <tt>wp</tt> to save the peaklist as <tt>hncoO1.peaks</tt>.
#Go to next step for [[XEASY Backbone Assignment|backbone assignment]]

==== '''Analysis of the (3,2)D GFT HNNCO spectrum''' ====

*Go to <tt>/protName/analysis/xeasy/hnco</tt>
*In UBNMR run macro <tt>make32DHncoPeak:</tt> listed as following to get (3,2)D GFT HNNCO peaklist. 
<blockquote><pre>init
read seq nhsqc.seq
read prot noe.prot

write prot hnco.prot
simulate 2D N+pC HN
simulate 2D N-pC HN
write peaks HNNCO.peaks
</pre></blockquote>
*Do the analysis in XEASY on the (3,2)D GFT HNNCO spectrum similar to that of 3D HNNCO spectrum.

 -- Main.GaohuaLiu - 16 Feb 2007</pre>

XEASY Introduction

2009-11-24T16:00:47Z

Jlmills:

XEASY [http://www.mol.biol.ethz.ch/groups/wuthrich_group/software http://www.mol.biol.ethz.ch/groups/wuthrich_group/software] is an X Window System based program for NMR spectrum analysis from the ETH.

 

*[[Media:Xeasy.doc|Xeasy.doc]]: A description on XEASY basics & assignment strategies.
*[[Media:MostlyUsedXEASYcommands.doc|MostlyUsedXEASYcommands.doc]]: An overview of the most commonly used XEASY commands.
*Three important files:
**[[XEASY Sequence List|Sequence List]]
**[[XEASY Atom List|Atom List]]
**[[XEASY Peak List|Peak List]]