Resonance Assignment/CARA/Aliphatic side-chain assignment: Difference between revisions

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== Introduction ==
 
 
 
== '''Aliphatic Side-chain Resonance Assignment''' ==
 
=== '''Introduction''' ===


For a short overview of heteronuclear side-chain assignment see this page:
For a short overview of heteronuclear side-chain assignment see this page:
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A combination of both approaches can be employed, however, assignment should be done as far as possible with the standard approach.
A combination of both approaches can be employed, however, assignment should be done as far as possible with the standard approach.


It is recommended to pick new spins in the "draft" mode first, so that a question mark "?" prefixes the actual spin label. Each spin system may have several spins with identical "draft" labels (e.g. <tt>?CG</tt>), though only one final label (<tt>CG</tt> in this example) is allowed. After you finish the assignment of a side chain, convert "draft" labels to permanent labels by right-clicking on a spin and selecting '''Accept Label'''. Using draft labels helps prevent the propagation of incorrect assignments in cases, when a particular challenging residue is skipped leaving a tentative assignment, which is not fixed at a later stage.
It is recommended to pick new spins in the "draft" mode first, so that a question mark "?" prefixes the actual spin label. Each spin system may have several spins with identical "draft" labels (e.g. ?CG), though only one final label (CG in this example) is allowed. After you finish the assignment of a side chain, convert "draft" labels to permanent labels by right-clicking on a spin and selecting '''Accept Label'''. Using draft labels helps prevent the propagation of incorrect assignments in cases, when a particular challenging residue is skipped leaving a tentative assignment, which is not fixed at a later stage.


When picking new spins attention should be paid to how the new chemical shift fits the statistical distribution for the corresponding spin type. Checking the '''View -> Show Rulers''' checkbox in SystemScope will display reference lines at average plus and minus four standard deviations in the strip panel. You can also consult the [http://www.bmrb.wisc.edu/ref_info/statsel.htm BMRB online chemical shift statistics page]. If you find a chemical shift that is more than 2 or 3 standard deviations away from average, it doesn't mean ''per se'' that the assignment is incorrect (for example, check out the maximum and minimum columns of the BMRB statistics). However, the probability of this really occurring is very low and, thus, it is a reason to double-check the assignments for this residue.
When picking new spins attention should be paid to how the new chemical shift fits the statistical distribution for the corresponding spin type. Checking the '''View -> Show Rulers''' checkbox in SystemScope will display reference lines at average plus and minus four standard deviations in the strip panel. You can also consult the [http://www.bmrb.wisc.edu/ref_info/statsel.htm BMRB online chemical shift statistics page]. If you find a chemical shift that is more than 2 or 3 standard deviations away from average, it doesn't mean ''per se'' that the assignment is incorrect (for example, check out the maximum and minimum columns of the BMRB statistics). However, the probability of this really occurring is very low and, thus, it is a reason to double-check the assignments for this residue.
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Also, note that the statistics can be misleading for diastereotopic protons and methyl groups of Val and Leu. Even though HB2 and HB3 of Phe have very close average values, in a real spectrum they can be far apart. The same is true for CG1/2 and HG1/2 of Val, as well as CD1/2 and HD1/2 of Leu.
Also, note that the statistics can be misleading for diastereotopic protons and methyl groups of Val and Leu. Even though HB2 and HB3 of Phe have very close average values, in a real spectrum they can be far apart. The same is true for CG1/2 and HG1/2 of Val, as well as CD1/2 and HD1/2 of Leu.


=== '''Displaying spectra in multiple windows'''  ===
== Displaying spectra with scopes ==


The complete set for aliphatic side chain assignment has 7 windows open at the same time:  
The complete set for aliphatic side chain assignment has 7 windows open at the same time:  
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Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.
Since it takes a lot of time to get all windows set up, avoid closing CARA. Fortunately it is quite stable and the Linux version, at least, can stay open for weeks.


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


To display a strip of interest in a StripScope window, select a residue from the pull-down menu. Spins belonging to the selected spins systems will appear in the panel below it. In the second panel below, a list of "strip anchors" (e.g. CA/HA and CB/HB pairs) will be displayed. Double-click on a spin pair, or right-click and select '''Show Strip'''. A corresponding strip will be displayed to the right. Its dimensions are '''X '''and'''Y''', as defined by the rotation of the spectrum.  
To display a strip of interest in a StripScope window, select a residue from the pull-down menu. Spins belonging to the selected spins systems will appear in the panel below it. In the second panel below, a list of "strip anchors" (e.g. CA/HA and CB/HB pairs) will be displayed. Double-click on a spin pair, or right-click and select '''Show Strip'''. A corresponding strip will be displayed to the right. Its dimensions are '''X '''and'''Y''', as defined by the rotation of the spectrum.  
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SystemScope in window 5 is used to verify whether diasterotopic spin pairs are degenerate, and pick a missing spin, if not. For example, you have HB/CB of Asn assigned. Display this HB/HB strip in window 5 and use right-click -&gt; '''Show Depth'''. In the depth plane zoom into the region around HB. If HB are non-degenerate and you have missed the other spin, you would see two strong HB2-HB3 peaks both symmetric with respect to diagonal. In addition, the two spin positions should exhibit very similar (though not necessarily identical) NOE peak patterns along Y, including HB-HA and HB-H peaks.
SystemScope in window 5 is used to verify whether diasterotopic spin pairs are degenerate, and pick a missing spin, if not. For example, you have HB/CB of Asn assigned. Display this HB/HB strip in window 5 and use right-click -&gt; '''Show Depth'''. In the depth plane zoom into the region around HB. If HB are non-degenerate and you have missed the other spin, you would see two strong HB2-HB3 peaks both symmetric with respect to diagonal. In addition, the two spin positions should exhibit very similar (though not necessarily identical) NOE peak patterns along Y, including HB-HA and HB-H peaks.


=== '''Calibration''' ===
After validating a few HA/CA and HB/CB chemical shifts you may want to adjust the calibration of 3D (H)CCH-COSY, 3D (H)CCH-TOCSY and 2D [13C, 1H] HSQCs. Since the final chemical shifts are used in 13C-resolved NOESY, calibration of other spectra must be adjusted to match it.
''expand with details''


 
== Validation of HA/CA and HB/CB assignments ==
=== '''Validation of HA/CA and HB/CB assignments''' ===


The first step before you actually pick new side chain spins is to verify that HA/CA assignments are correct and adjust them, if needed.
The first step before you actually pick new side chain spins is to verify that HA/CA assignments are correct and adjust them, if needed.
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Keep in mind that HA and HB assignments originated from the indirect 1H dimension of 3D HBHA(CO)NH. The direct 1H dimension in (H)CCH-COSY, (H)CCH-TOCSY and 13C-resolved NOESY offers a much higher resolution. Therefore, some HB2 and HB3 spins (or HA2 and HA3 for Gly), which previously were considered degenerate and picked as HB (or HA for Gly), may be resolved in these spectra. If this happens, HB2 and HB3 (or HA2 and HA3) have to be picked separately. For example, you can change the HB label to HB2, and pick HB3 as a new spin.
Keep in mind that HA and HB assignments originated from the indirect 1H dimension of 3D HBHA(CO)NH. The direct 1H dimension in (H)CCH-COSY, (H)CCH-TOCSY and 13C-resolved NOESY offers a much higher resolution. Therefore, some HB2 and HB3 spins (or HA2 and HA3 for Gly), which previously were considered degenerate and picked as HB (or HA for Gly), may be resolved in these spectra. If this happens, HB2 and HB3 (or HA2 and HA3) have to be picked separately. For example, you can change the HB label to HB2, and pick HB3 as a new spin.


=== '''Common validation issues''' ===
After validating a few HA/CA and HB/CB chemical shifts you may want to adjust the calibration of 3D (H)CCH-COSY, 3D (H)CCH-TOCSY and 2D [13C, 1H] HSQCs. Since the final chemical shifts are used in 13C-resolved NOESY, calibration of other spectra must be adjusted to match it.
 
== Common validation issues ==


==== '''Proximity to water resonance''' ====
*Proximity to water resonance


The chemical shift of H2O at 15 &deg;C is about 4.75 ppm. HA resonances of all residue types as well as HB lines of Ser and Thr can be very close to this position. If that happens, either the entire strip may be missing (as water line is often computationally subtracted from the spectrum during processing) or the peaks may be masked by the strong local noise. When the former is true, precise HA position may only be taken from the indirect dimension of the H-N strip of 15N-resolved NOESY or the HB-CB and other strips of the aliphatic 13C-resolved NOESY.
The chemical shift of H2O at 15 &deg;C is about 4.75 ppm. HA resonances of all residue types as well as HB lines of Ser and Thr can be very close to this position. If that happens, either the entire strip may be missing (as water line is often computationally subtracted from the spectrum during processing) or the peaks may be masked by the strong local noise. When the former is true, precise HA position may only be taken from the indirect dimension of the H-N strip of 15N-resolved NOESY or the HB-CB and other strips of the aliphatic 13C-resolved NOESY.


==== '''Missing assignments''' ====
*Missing assignments


For sensitivity-limited samples it is common that some backbone assignments will be missing.
For sensitivity-limited samples it is common that some backbone assignments will be missing.
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If more spins are missing, and you don't have a starting strip in the (H)CCH-COSY, it is recommended to skip this side-chain. It can be assigned using alternative approaches after all "easy" side chains are assigned.
If more spins are missing, and you don't have a starting strip in the (H)CCH-COSY, it is recommended to skip this side-chain. It can be assigned using alternative approaches after all "easy" side chains are assigned.


==== '''Low sensitivity''' ====
*Low sensitivity


Certain residues may be exchange-broadened. It is better to skip them first and try to assign them after most of the remaining side chains are assigned.
Certain residues may be exchange-broadened. It is better to skip them first and try to assign them after most of the remaining side chains are assigned.
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If you have consistent sensitivity issues with a lot of residues, you probably need to acquire longer (H)CCH and/or NOESY spectra.
If you have consistent sensitivity issues with a lot of residues, you probably need to acquire longer (H)CCH and/or NOESY spectra.


==== '''Resonance overlap''' ====
*Resonance overlap


Here more attention should be payed to the shape of the peaks and small difference in peak centers between close strips.
Here more attention should be payed to the shape of the peaks and small difference in peak centers between close strips.


==== '''Incorrect assignments''' ====
*Incorrect assignments


If you don't see the expected patterns in (H)CCH-COSY and 13C-resolved NOESY, and you don't have sensitivity or water resonance issues - then it is likely that the HA/CA and/or HB/CB assignments at a previous stage were incorrect.
If you don't see the expected patterns in (H)CCH-COSY and 13C-resolved NOESY, and you don't have sensitivity or water resonance issues - then it is likely that the HA/CA and/or HB/CB assignments at a previous stage were incorrect.
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# Pick the alternative location in HBHA(CO)NH or 15N-resolved NOESY
# Pick the alternative location in HBHA(CO)NH or 15N-resolved NOESY


A very common situation is when two spins systems overlap in the H-N plane and, while CA and CB assignments are correct, their HA and HB assignments are inadvertently swapped. Look carefully at the HBHA(CO)NH and HNCA or HNCACB in window 7.
A very common situation is when two spins systems overlap in the H-N plane and, while CA and CB assignments are correct, their HA and HB assignments are inadvertently swapped. Look carefully at the HBHA(CO)NH and HNCA or HNCACB in window 7. Once again, if you cannot fix this problem easily it is better to leave it until most residues are assigned.


Once again, if you can fix this problem easily it is better to leave it until most residues are assigned.
== Assignment strategies for individual residue types ==


(''add more info and make it readble'')
=== Gly ===


=== '''Standard side-chain assignment strategies for individual residue types''' ===
Since technically Gly does not have a side chain, aliphatic assignment is limited to validation of HA2/CA and HA3/CA assignments (or HA/CA, if degenerate).
 
==== '''Gly''' ====
 
Side-chain assignment for Gly is limited to validation of HA2/CA and HA3/CA assignments (or HA/CA, if degenerate).


Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA2 and H-HA3 intra peaks, as well as HA2(i)-H(i+1) and HA3(i)-H(i+1) sequential peaks.
Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA2 and H-HA3 intra peaks, as well as HA2(i)-H(i+1) and HA3(i)-H(i+1) sequential peaks.
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Note that the chemical shifts of HA2 and HA3 are typically very different. Degenerate HA spins are rare, but not uncommon.
Note that the chemical shifts of HA2 and HA3 are typically very different. Degenerate HA spins are rare, but not uncommon.


==== '''Ala, Asn, Asp, Cys, His, Phe, Ser, Trp and Tyr''' ====
=== Ala, Asn, Asp, Cys, His, Phe, Ser, Trp and Tyr ===


These residues do not have aliphatic H/C moieties beyond HB/CB. Therefore, side-chain assignment is complete after validation of HA/CA and HB/CB assignments.
These residues do not have aliphatic H/C moieties beyond HB/CB. Therefore, side-chain assignment is complete after validation of HA/CA and HB/CB assignments.
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It may happen that CA and CB chemical shifts overlap in Ser. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Thr.
It may happen that CA and CB chemical shifts overlap in Ser. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Thr.


==== '''Gln, Glu and Met''' ====
=== Gln, Glu and Met ===


For these residues you need to assign HG2/CG and HG3/CG (or HG/CG, if degenerate). Follow this procedure:
For these residues you need to assign HG2/CG and HG3/CG (or HG/CG, if degenerate). Follow this procedure:
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The HE/CE methyl group of Met is assigned at later stage.
The HE/CE methyl group of Met is assigned at later stage.


==== '''Arg and Pro''' ====
=== Arg and Pro ===


For these residues you need to assign HG2/CG, HG3/CG (or HG/CG, if degenerate), HD2/CD and HD3/CD (or HD/CD, if degenerate).  
For these residues you need to assign HG2/CG, HG3/CG (or HG/CG, if degenerate), HD2/CD and HD3/CD (or HD/CD, if degenerate).  
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In both Arg and Pro HG can overlap with HB.
In both Arg and Pro HG can overlap with HB.


==== '''Lys''' ====
=== Lys ===


For Lys you need to assign HG2/CG, HG3/CG (or HG/CG), HD2/CD and HD3/CD (or HD/CD), and HE2/CE and HE3/CE (or HE/CE)
For Lys you need to assign HG2/CG, HG3/CG (or HG/CG), HD2/CD and HD3/CD (or HD/CD), and HE2/CE and HE3/CE (or HE/CE)
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Also, HE/CE pairs of different Lys residues tend to be very close together, and cannot be told apart in 2D [13C, 1H] HSQC.
Also, HE/CE pairs of different Lys residues tend to be very close together, and cannot be told apart in 2D [13C, 1H] HSQC.


==== '''Thr''' ====
=== Thr ===


Thr can be assigned according to procedure for Glu, Gln and Met, though Thr only has one HG2, and the carbon is labeled CG2 (not CG).
Thr can be assigned according to procedure for Glu, Gln and Met, though Thr only has one HG2, and the carbon is labeled CG2 (not CG).
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It may happen that CA and CB chemical shifts overlap in Thr. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Ser.
It may happen that CA and CB chemical shifts overlap in Thr. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Ser.


==== '''Val''' ====
=== Val ===


For Val you have to assign both HG1/CG1 and HG2/CG2 methyl groups.
For Val you have to assign both HG1/CG1 and HG2/CG2 methyl groups.
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Another issue may be the degeneracy of HG1 and HG2, when CG1/CG2 are not degenerate. In this case, too, four spins (CG1, CG2, HG1 and HG2) should be picked.
Another issue may be the degeneracy of HG1 and HG2, when CG1/CG2 are not degenerate. In this case, too, four spins (CG1, CG2, HG1 and HG2) should be picked.


==== '''Leu''' ====
=== Leu ===


For Leu you need to assign HG/CG, HD1/CD1 and HD2/CD2 groups.
For Leu you need to assign HG/CG, HD1/CD1 and HD2/CD2 groups.
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Note that HB2 and HB3 are seldom degenerate in Leu.
Note that HB2 and HB3 are seldom degenerate in Leu.


==== '''Ile''' ====
=== Ile ===


Despite the complexity of its branched side-chain, Ile is probably the easiest to assign due to all its aliphatic 13C and 1H spins having distinct chemical shifts.
Despite the complexity of its branched side-chain, Ile is probably the easiest to assign due to all its aliphatic 13C and 1H spins having distinct chemical shifts.
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HB can overlap with either HG12 or HG13
HB can overlap with either HG12 or HG13


=== '''Filling in the gaps''' ===
== Filling in the gaps ==
 
After the first pass through the sequence some residues that were skipped. You may also have small gaps in the backbone assignment.
 
One category of skipped residues are those directly preceding prolines, since they will have CA and CB assignments at most. According to the alternative approach you have to find HA and HB in the H strip is the same residue in 15N-resolved NOESY (windows 6 and 7) and in the HD2 and HD3 strips of the succeeding Pro in 13C-resolved NOESY (window 3). Note that the latter is only valid for ''trans'' peptide bond. You should consider looking at the HA strip of Pro, if you don't any candidates in HD2 and HD3 strips.
 
 
----------------------------------------------------------------------------------------------------------
 
 
(''rewrite below'')
 
The best way to assign side chains is to open HCCH-COSY (central peak and two basic spectra) and aliphatic 13C-resolved NOESY (or the corresponding part of the simultaneous NOESY) in SystemScope windows and to use them simultaneously to assign one residue at a time. (H)CCH-TOCSY can be used instead or in addition to (H)CCH-COSY.
 
(H)CCH-COSY and (H)CCH-TOCSY spectra should be opened with '''Open SystemScope (rotated)...'''. A rotation matrix window will pop up. Choose the following orientation: Hinept dimension must be Z, Ccosy must be Y, and Cinept must be X. Thus, the left panel of SystemScope will contain a C-C plane (with C-cosy dimension vertical), and the right panel will contain the H-C plane. This issue is addressed in detail in this FAQ question: http://www.cara.ethz.ch/Wiki/FAQ#III10
 
 
13C-resolved NOESY should be opened in the default orientation. Open C13 HSQC in the PolyScope, the SimNOESY being loaded in the strips. The SimNOESY Cali is also loaded in two different SystemScope windows, with the normal orientation in one and another rotated with the Hinept in the Z, Hnoesy along Y and Cali along X.
 
Normally you will have HA/CA and HB/CB anchors for each residue from the previous assignment stage. That means that the side-chain assignment is already essentially complete for AMX spin systems. Only verification and fine adjustments of 13C and 1H shifts need to be done in the aliphatic 13C-resolved NOESY.
 
For longer side-chains you can try mixing the following approaches.
 
* (H)CCH-first approach:
** In (H)CCH-COSY or (H)CCH-TOCSY pick the CG spins in the left plane
** In (H)CCH-COSY or (H)CCH-TOCSY use '''Show Orthogonal''' to display the H-CG plane on the right
** In (H)CCH-COSY or (H)CCH-TOCSY pick HG spins in the right plane
** Verify HG and CG spins with aliphatic 13C-resolved NOESY
** Repeat these steps for other spins up the side-chain...
 
* (4,3)D-HCCH approach:
** Run '''GFT_HCCH_Calc''' Lua script to simulate side-chain ?C*+/-H* combinations.
** Move side-chain ?CGpHG and ?CGmHG spins to their correct positions.
** Select '''Accept Label''' to drop the draft designation (remove =?=)
** Run '''GFT_HCCH_Calc''' Lua script again to update CG and HG shifts.
** Verify HG and CG in (4,3)D-HCCH (central peak subspectrum - same as (H)CCH-COSY) and aliphatic 13C-resolved NOESY
** Repeat these steps for other spins up the side-chain...
 
* NOESY-first approach:
** In 13C-resolved (or 15N resolved) NOESY pick HG spin candidates
** In aliphatic 13C-resolved NOESY use '''Show Orthogonal''' to display the HG-CG plane on the right
** In aliphatic 13C-resolved NOESY pick the CG spin (right plane in SystemScope)
** Verify HG and CG spins with aliphatic (H)CCH-COSY
** Repeat these steps for other spins up the side-chain...
 
You can use temporary spin assignments by putting a question mark (=?=) as the first character of a spin label. Such spin labels are also called "draft" labels.
 
'''GFT_HCCH_Calc''' work by using spins with "accepted" labels (that is, without question marks) to generate missing spins as "drafts" (with question marks). If no "accepted" spins are present, random-coil chemical shifts are used. For example:
* if you have HG and CG spins assigned, then ?CGpHG and ?CGmHG spins will be created.
* if CGpHG and CGmHG spins exist, then ?CG and ?HG spins will be created.
* if CGpHG, CGmHG, HG and CG exist, then their shifts will be checked for consistency.
* if no HG, CG, CGpHG and CGmHG exist where they should, then ?HG, ?CG, ?CGpHG and ?CGmHG spins will be created.


'''IMPORTANT!''' In the end you would want the "true" (non-GFT spins) to match the aliphatic 13C-resolved NOESY spectrum. Therefore, use 13C-resolved NOESY spectrum to finalize side-chain assignment and fine-tune spin positions. If there is a mismatch between NOESY and HCCH spectra, use spin aliases in (H)CCH only.
After the first pass through the sequence some residues were skipped. You may also have small gaps in the backbone assignment.


'''IMPORTANT!''' If you have Leu or Val with completely degenerate methyl groups (both 13C and 1H shifts are the same), you should ultimately label them as CD1/HD and CG1/HG, respectively. Even though in this case CD and CG are valid labels in CARA (as 13C atom groups aka. pseudoatoms), they are not compatible with CYANA 2.1. Also see the related issue with CD and CE spins of Tyr and Phe.
One category of skipped residues are those directly preceding prolines, since they will have CA and CB assignments at most. According to the alternative approach you have to find HA and HB in the H strip is the same residue in 15N-resolved NOESY (windows 6 and 7) and in the HD2 and HD3 strips of the succeeding Pro in 13C-resolved NOESY (window 3). Note that the latter is only valid for ''trans'' peptide bond. You should consider looking at the HA strip of Pro, if you don't see any candidates in HD2 and HD3 strips.


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

Latest revision as of 14:21, 31 August 2012

Introduction

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

At this point you should have a nearly complete backbone assignment. Most residues should have CA and CB resonances assigned in HNCA, HNCACB, CBCA(CO)NH or CA(CO)NH and HA and HB resonances assigned with HBHA(CO)NH. This topic describes aliphatic side-chain assignment using 3D (H)CCH-COSY, 3D (H)CCH-TOCSY (optional) and 13C-resolved NOESY.

The main steps in aliphatic side-chain assignment are:

  • Validation of HA/CA and HB/CB assignments
  • In some cases, assignment of HA/CA or HB/CB spins (if unassigned previously)
  • Assignment of spins further up the side-chain (HG/CG, HD/CD and HE/CE)
  • Precise positioning of spins in the 13C-resolved NOESY

The recommended procedure is to try to assign one residue at a time as one goes along the protein sequence. One should pick the low-hanging fruits first, though, by looking first at the residues with both HA/CA and HB/CB anchors and considering the rest in the second pass. Also, skip the residues, for which you are not sure how to complete their assignment. The risk of sticking to an incorrect assignment is thus minimized. Once you have a nearly complete assignment, you can even try to locate the missing spins among unassigned spins in 2D [13C, 1H]-HSQC.

There are two approaches to assigning a side chain:

  • The standard approach is to pick 1H and 13C spins in (H)CCH-COSY or (H)CCH-TOCSY and verify them against 13C-resolved NOESY while progressing up the side chain from CB/HB to CG/HG to CD/HD to CE/HE. (H)CCH-TOCSY also allows to hop over a 13C spin, for example, going from HB/CB to HD/CD, while skipping HG/CG. This approach reduces the risk of incorrect assignments because spins are picked in through-bond correlation experiments.
  • The alternative approach is to pick spins 1H in 13C- or 15N-resolved NOESY and 13C spins in 13C-resolved NOESY and verify them against (H)CCH-COSY and (H)CCH-TOCSY. This approach carries a much higher risk of incorrect assignments and should in principle be used only if the standard approach fails.

A combination of both approaches can be employed, however, assignment should be done as far as possible with the standard approach.

It is recommended to pick new spins in the "draft" mode first, so that a question mark "?" prefixes the actual spin label. Each spin system may have several spins with identical "draft" labels (e.g. ?CG), though only one final label (CG in this example) is allowed. After you finish the assignment of a side chain, convert "draft" labels to permanent labels by right-clicking on a spin and selecting Accept Label. Using draft labels helps prevent the propagation of incorrect assignments in cases, when a particular challenging residue is skipped leaving a tentative assignment, which is not fixed at a later stage.

When picking new spins attention should be paid to how the new chemical shift fits the statistical distribution for the corresponding spin type. Checking the View -> Show Rulers checkbox in SystemScope will display reference lines at average plus and minus four standard deviations in the strip panel. You can also consult the BMRB online chemical shift statistics page. If you find a chemical shift that is more than 2 or 3 standard deviations away from average, it doesn't mean per se that the assignment is incorrect (for example, check out the maximum and minimum columns of the BMRB statistics). However, the probability of this really occurring is very low and, thus, it is a reason to double-check the assignments for this residue.

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

Displaying spectra with scopes

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

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

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

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

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

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

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

Working with SystemScope

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

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

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

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

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


Validation of HA/CA and HB/CB assignments

The first step before you actually pick new side chain spins is to verify that HA/CA assignments are correct and adjust them, if needed.

  1. In windows 1 ((H)CCH-COSY) and 3 (13C-resolved NOESY) display the HA/CA strip. For Gly residues having two strips, HA2/CA and HA3/CA, repeat for each.
  2. Verify that in window 1 you see two peaks in the strip, CA and CB (Gly residues will only have the CA diagonal peak).
  3. In window 3 you should see characteristic intra-residue H-HA and HA-HB and sequential HA(i)-H(i+1) NOE peaks.
  4. For Gly in window 5 verify whether you have degenerate HA or non-degenerate HA2 and HA3.
  5. In the HSQC plane of window 3 move the HA/CA (or HA2/CA and HA3/CA of Gly) peaks so that the NOESY strips are centered.
  6. In windows 1 and 3 display the HB/CB strip. Many residues will have two strips, HB2/CB and HB3/CB.
  7. Verify that in window 1 you see two or more peaks in the strip - CA, CB and one or two CG peaks for long side chains.
  8. In window 3 you should see characteristic intra-residue NOE peaks to HA-HB and, possibly, H-HB.
  9. In window 5 verify whether you have degenerate HB or non-degenerate HB2 and HB3.
  10. In the HSQC plane of window 3 move the HB/CB (or HB2/CB and HB3/CB) peaks so that the NOESY strips are centered.

Ideally, all CA, CB, HA (or HA2 an HA3), HB (or HB2 and HB3) should be present and their chemical shifts should match (H)CCH-COSY, (H)CCH-TOCSY and 13C-resolved NOESY quite well.

Keep in mind that HA and HB assignments originated from the indirect 1H dimension of 3D HBHA(CO)NH. The direct 1H dimension in (H)CCH-COSY, (H)CCH-TOCSY and 13C-resolved NOESY offers a much higher resolution. Therefore, some HB2 and HB3 spins (or HA2 and HA3 for Gly), which previously were considered degenerate and picked as HB (or HA for Gly), may be resolved in these spectra. If this happens, HB2 and HB3 (or HA2 and HA3) have to be picked separately. For example, you can change the HB label to HB2, and pick HB3 as a new spin.

After validating a few HA/CA and HB/CB chemical shifts you may want to adjust the calibration of 3D (H)CCH-COSY, 3D (H)CCH-TOCSY and 2D [13C, 1H] HSQCs. Since the final chemical shifts are used in 13C-resolved NOESY, calibration of other spectra must be adjusted to match it.

Common validation issues

  • Proximity to water resonance

The chemical shift of H2O at 15 °C is about 4.75 ppm. HA resonances of all residue types as well as HB lines of Ser and Thr can be very close to this position. If that happens, either the entire strip may be missing (as water line is often computationally subtracted from the spectrum during processing) or the peaks may be masked by the strong local noise. When the former is true, precise HA position may only be taken from the indirect dimension of the H-N strip of 15N-resolved NOESY or the HB-CB and other strips of the aliphatic 13C-resolved NOESY.

  • Missing assignments

For sensitivity-limited samples it is common that some backbone assignments will be missing.

HA, CA and CB assignments present - HB is missing:

  1. Select the HA/CA strip in 3D (H)CCH-COSY (window 1).
  2. Select the CB spin, right-click and select Show Orthogonal to display the orthogonal plane.
  3. Pick HB2 and HB3 (or HB) spins in the orthogonal plane. The correct HB spin position should exhibit CA and CB peaks.
  4. Open the new HB2/CB and HB3/CB (or HB/CB) strips in window 3. Verify, that HA-HB (likely H-HB) NOE cross-peaks are present. Adjust the HB/CB positions according to aliphatic 13C-resolved NOESY.
  5. Optionally, verify assignment by checking HA/CA and HB/CB strips in 3D (H)CCH-TOCSY (window 2)
  6. Verify the new HB assignments in HBHA(CO)NH (window 7).

HA, CA and HB assignments present - CB is missing:

  1. Select the HA/CA strip in 3D (H)CCH-COSY (window 1)
  2. Pick the CB spin in the strip panel of window 1
  3. Select the HB/CB strip(s) in window 1 and use Show Depth. Verify that CA and CB cross-peaks are present
  4. Open the new HB2/CB and HB3/CB (or HB/CB) strips in window 3. Verify, that HA-HB (likely H-HB) NOE cross-peaks are present. Adjust the HB/CB positions according to aliphatic 13C-resolved NOESY.
  5. Optionally, verify assignment by checking HA/CA and HB/CB strips in 3D (H)CCH-TOCSY (window 2).
  6. Verify the new CB assignments in HNCACB and CBCA(CO)NH (window 7).

HA and CA are present - CB and HB are missing:

  1. Select the HA/CA strip in 3D (H)CCH-COSY (window 1).
  2. Pick the CB spin in window 1
  3. Select the CB spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HB spin(s) in the orthogonal plane. The correct HB spin position should exhibit CA and CB peaks.
  5. Open the new HB/CB strip(s) in window 3. Verify, that HA-HB, (possibly H-HB) NOE patterns are present. Adjust the HB/CB position according to aliphatic 13C-resolved NOESY.
  6. Optionally, verify assignment by checking all strips in 3D (H)CCH-TOCSY (window 2)
  7. Verify the new CB assignments in HNCACB and CBCA(CO)NH (window 7).
  8. Verify the new HB assignments in HBHA(CO)NH (window 7).

HB (or HB2 and HB3) and CB are present - CA and HA are missing:

  1. Select an HB/CB strip in window 1.
  2. Pick the CA spin in window 1.
  3. Select the CA spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick the HA spin in the orthogonal plane. The correct HA spin position should exhibit CB and CA peaks.
  5. Open the new HA/CA strip in window 3. Verify, that HA-HB and H-HA NOE cross-peaks are present. Adjust the HA/CA position according to aliphatic 13C-resolved NOESY.
  6. Optionally, Verify assignment by checking all strips in 3D (H)CCH-TOCSY
  7. Verify the new CA assignments in HNCACB and CBCA(CO)NH (window 7).
  8. Verify the new HA assignments in HBHA(CO)NH (window 7).


If more spins are missing, and you don't have a starting strip in the (H)CCH-COSY, it is recommended to skip this side-chain. It can be assigned using alternative approaches after all "easy" side chains are assigned.

  • Low sensitivity

Certain residues may be exchange-broadened. It is better to skip them first and try to assign them after most of the remaining side chains are assigned.

If you have consistent sensitivity issues with a lot of residues, you probably need to acquire longer (H)CCH and/or NOESY spectra.

  • Resonance overlap

Here more attention should be payed to the shape of the peaks and small difference in peak centers between close strips.

  • Incorrect assignments

If you don't see the expected patterns in (H)CCH-COSY and 13C-resolved NOESY, and you don't have sensitivity or water resonance issues - then it is likely that the HA/CA and/or HB/CB assignments at a previous stage were incorrect.

See if you are missing peaks in HA/CA strip, HB/CB strip or both. It is unlikely that CA or CB assignment are wrong, since this would have interfered with spin system linking and matching during backbone assignment.

  1. Find the correct HA or HB by locating the expected pattern in the depth plane (right click -> Show Depth). Use right-click -> Move Orthogonal Spin.
  2. Pick the alternative location in HBHA(CO)NH or 15N-resolved NOESY

A very common situation is when two spins systems overlap in the H-N plane and, while CA and CB assignments are correct, their HA and HB assignments are inadvertently swapped. Look carefully at the HBHA(CO)NH and HNCA or HNCACB in window 7. Once again, if you cannot fix this problem easily it is better to leave it until most residues are assigned.

Assignment strategies for individual residue types

Gly

Since technically Gly does not have a side chain, aliphatic assignment is limited to validation of HA2/CA and HA3/CA assignments (or HA/CA, if degenerate).

Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA2 and H-HA3 intra peaks, as well as HA2(i)-H(i+1) and HA3(i)-H(i+1) sequential peaks.

Note that the chemical shifts of HA2 and HA3 are typically very different. Degenerate HA spins are rare, but not uncommon.

Ala, Asn, Asp, Cys, His, Phe, Ser, Trp and Tyr

These residues do not have aliphatic H/C moieties beyond HB/CB. Therefore, side-chain assignment is complete after validation of HA/CA and HB/CB assignments.

Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA and H-HB intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1) sequential peaks.

Note that the chemical shifts of HB2 and HB3 of Phe and Tyr (and to a lesser extent of His and Trp) are frequently very different.

It may happen that CA and CB chemical shifts overlap in Ser. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Thr.

Gln, Glu and Met

For these residues you need to assign HG2/CG and HG3/CG (or HG/CG, if degenerate). Follow this procedure:

  1. Select an HB/CB strip in window 1
  2. Pick CG spin(s) in window 1
  3. Select a CG spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HG spin(s) in the orthogonal plane. The correct HG spin position should exhibit CB and CG peaks.
  5. Open the new HG/CG strip(s) in window 3. Verify, that HA-HG, HB-HG (possibly H-HG) NOE patterns are present. Adjust the HG/CG position according to aliphatic 13C-resolved NOESY.
  6. In window 5 verify whether HG assignments are degenerate.
  7. Verify assignment by checking all strips in 3D (H)CCH-TOCSY.
  8. Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA and H-HB, possibly H-HG intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1) and HG(i)-H(i+1) sequential peaks.


The assignment can be complicated by the fact that these residues have very low chemical shift dispersion of HB/CB and HG/CG pairs. It is common for HB/CB and HG/CG peaks from several Gln and Glu residues in a protein to form a single blob. In addition, HB and HG chemical shifts within a single residue are usually very similar.

If you cannot reliably pick the CG spin in HB/CB strip of 3D (H)CCH-COSY (due to overlap, for example), try locating it in the HA/CA 3D (H)CCH-TOCSY.

If that fails, try the alternative approach:

  1. Locate HG candidates in the HB/CB and HA/CA strips of aliphatic 3D C13-resolved NOESY (window 3), or H/N strip of 3D N15-resolved NOESY (window 5).
  2. In window 4 select an HG candidate and use Show Orthogonal to display the orthogonal plane.
  3. In the orthogonal plane locate the CG candidate. The correct CG position should exhibit HG-HB, HG-HA, possibly HG-H NOE cross-peaks. If there are no good CG choices, repeat from step 2 with the next HG candidate.
  4. Validate and adjust positions of HG/CG strip in window 3.

The HE/CE methyl group of Met is assigned at later stage.

Arg and Pro

For these residues you need to assign HG2/CG, HG3/CG (or HG/CG, if degenerate), HD2/CD and HD3/CD (or HD/CD, if degenerate).

HG/CG should be assigned in the same manner as Glu, Gln and Met. For HD/CD follow this procedure:

  1. Select an HG/CG strip in window 1.
  2. Pick CD spin(s) in window 1.
  3. Select a CD spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HD spin(s) in the orthogonal plane. The correct HD spin position should exhibit CG and CD peaks
  5. Open the new HD/CD strip(s) in window 3. Verify, that HG-HD NOE patterns are present. Adjust the HD/CD position according to aliphatic 13C-resolved NOESY.
  6. Verify HD assignments in window 5.
  7. Verify assignment by checking all strips in 3D (H)CCH-TOCSY (window 2).
    1. For Arg, validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA and H-HB, possibly H-HG intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1) and HG(i)-H(i+1) sequential peaks.
    2. For Pro, validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe HA(i)-H(i+1) and, possibly, HB(i)-H(i+1) sequential peaks. In window 3, verify that you observe HA(i-1)-HD2(i) and HA(i-1)-HD3(i) sequential NOE peaks for trans Xxx-Pro bond, or HA(i-1)-HA(i) sequential NOE peaks for cis Xxx-Pro bond.

Pro residues rarely have degenerate HB, HG or HD, though sometimes they happen. HD2/CD and HD3/CD cross-peaks can be guessed from 2D aliphatic [13C, 1H] HSQC, as they exclusively occupy a distinct region around (3.6 ppm, 50 ppm). HD2 and HD3 of a particular Pro residue can be picked from HA(i-1)-HD2(i) and HA(i-1)-HD3(i) NOE crosspeaks off the HA of a previous residue (this is true only for the most common trans Xxx-Pro bond, for rare cis Xxx-Pro peptide bonds these peaks are absent). The side-chain assignment can then proceed "backwards" toward HA/CA.

In both Arg and Pro HG can overlap with HB.

Lys

For Lys you need to assign HG2/CG, HG3/CG (or HG/CG), HD2/CD and HD3/CD (or HD/CD), and HE2/CE and HE3/CE (or HE/CE)

HG/CG and HD/CD are assigned in the same manner as Arg and Pro. For HE/CE follow this procedure:

  1. Select an HD/CD strip in window 1.
  2. Pick CE spin(s) in window 1.
  3. Select a CE spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HE spin(s) in the orthogonal plane. The correct HE spin position should exhibit CD and CE peaks.
  5. Open the new HE/CE strip(s) in window 3*. Verify, that HD-HE NOE patterns are present. Adjust the HE/CE position according to aliphatic 13C-resolved NOESY.
  6. In window 5 verify whether HE2 and HE3 are degenerate.
  7. Verify assignment by checking all strips in 3D (H)CCH-TOCSY.
  8. Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA and H-HB, possibly H-HG intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1) and HG(i)-H(i+1) sequential peaks.


Chemical shifts of HB, HG and HD of Lys are typically very similar, which complicates validation with 13C-resolved NOESY. For example, the HG-HD cross-peaks are often very close to the diagonal. In addition to that, Lys residues are frequently located on the protein surface and their side-chains exhibit only intra-residue NOEs. Thus, HD assignments can often only be validated through the HD-HE cross-peaks, since HE have distinct chemical shifts.

Also, HE/CE pairs of different Lys residues tend to be very close together, and cannot be told apart in 2D [13C, 1H] HSQC.

Thr

Thr can be assigned according to procedure for Glu, Gln and Met, though Thr only has one HG2, and the carbon is labeled CG2 (not CG).

Thr is the only residue whose HB can be found either upfield or downfield of HA - it can also overlap with HA. Both can be in proximity to the water resonance, complicating assignment.

It may happen that CA and CB chemical shifts overlap in Thr. Thus the expected patterns in (H)CCH-COSY and (H)CCH-TOCSY will be absent. The same is also true for Ser.

Val

For Val you have to assign both HG1/CG1 and HG2/CG2 methyl groups.

  1. Select the HB/CB strip in window 1.
  2. Pick CG1 and CG2 spins in window 1. If only one peak is visible, pick it as CG1 (for compatibility with CYANA).
  3. Select CG1 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HG1 spin in the orthogonal plane. The correct HG1 spin position should exhibit CB and CG1 peaks.
  5. Select CG2 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  6. Pick HG2 spin in the orthogonal plane. The correct HG2 spin position should exhibit CB and CG2 peaks.
  7. Open the new HG1/CG1 strip in window 3. Verify, that HA-HG1, HB-HG1, HG2-HG1 (possibly H-HG) NOE patterns are present. Adjust the HG1/CG1 position according to aliphatic 13C-resolved NOESY.
  8. Repeat the last step for the HG2/CG2 pair.
  9. Verify assignment by checking all strips in 3D (H)CCH-TOCSY
  10. Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA, H-HB, H-HG1 and H-HG2 intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1), HG1(i)-H(i+1), HG2(i)-H(i+1) sequential peaks.


There may be difficult cases when only one CG peak is visible instead of two. There are two possibilities:

  • It is very rare that both CG1/CG2 and HG1/HG2 spins are degenerate. One can tell that this is the case if there is only one HG candidate in the orthogonal plane. Only two spins should be assigned - CG1 and HG (note that CG label is not compatible with CYANA and should not be used).
  • A more common occurrence is when only CG1/CG2 are degenerate and HG1/HG2 are not. If this is the case, the orthogonal plane for CG1 and CG2 will apparently contain two valid HG1 and HG2 candidates. Four spins (CG1, CG2, HG1 and HG2) should be picked even though CG1 and CG2 shifts may be identical. The methyl groups can be later distinguished for stereospecific assignment.

Another issue may be the degeneracy of HG1 and HG2, when CG1/CG2 are not degenerate. In this case, too, four spins (CG1, CG2, HG1 and HG2) should be picked.

Leu

For Leu you need to assign HG/CG, HD1/CD1 and HD2/CD2 groups.

  1. Select an HB/CB strip in window 1.
  2. Pick CG spin in window 1.
  3. Select the CG spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick HG spin in the orthogonal plane. The correct HG spin position should exhibit CB and CG peaks (plus unassigned CD1 and CD2 peaks).
  5. Open the new HG/CG strip in window 3. Verify, that HB-HG (possibly HB-HG and H-HG) NOE patterns are present. Adjust the HG/CG position according to aliphatic 13C-resolved NOESY.
  6. Select the HG/CG strip in window 1.
  7. Pick CD1 and CD2 spins in window 1. If only one peak is visible, pick it as CD1 (for compatibility with CYANA).
  8. Select CD1 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  9. Pick HD1 spin in the orthogonal plane. The correct HD1 spin position should exhibit CG and CD1 peaks.
  10. Select CD2 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  11. Pick HD2 spin in the orthogonal plane. The correct HD2 spin position should exhibit CG and CD2 peaks.
  12. Open the new HD1/CD1 strip in window 3. Verify that HB-HD1, HG-HD1, HD2-HD1 (possibly HA-HD1) NOE patterns are present. Adjust the HD1/CD1 position according to aliphatic 13C-resolved NOESY.
  13. Repeat the last step for the HD2/CD2 pair.
  14. Verify assignment by checking all strips in 3D (H)CCH-TOCSY
  15. Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA, H-HB, possibly H-HG, H-HD1 and H-HD2 intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1), HG(i)-H(i+1), HD1(i)-H(i+1), HD2(i)-H(i+1) sequential peaks.


There my be degeneracy issues with HD1, HD2, CD1 and CD2 spins, which are the same as those of HG1, HG2, CG1 and CG2 spins of Val above.

Additional problem could be the overlap of CG and CD1 (or, equivalently, CG and CD2). In this case the orthogonal plane should contain both HG and HD1 (or HG and HD2) candidates, which typically have distinct chemical shifts.

Another issue is low intensity of the HG/CG strip, which often complicates HG assignment. An additional hindrance is that HG often overlaps with HB2 or HB3. To overcome this, you can hop over to CD1 and CD2 by picking them from HB2/CB and HB3/CB strips in 3D (H)CCH-TOCSY. HG can the picked using the alternative approached by looking for HG-HD1 and HG-HD2 cross-peaks in 13C-resolved NOESY.

Note that HB2 and HB3 are seldom degenerate in Leu.

Ile

Despite the complexity of its branched side-chain, Ile is probably the easiest to assign due to all its aliphatic 13C and 1H spins having distinct chemical shifts.

In Ile you have to assign HG12/CG, HG13/CG, HG2/CG2 and HD1/CD1 spins

  1. Select the HB/CB strip in window 1.
  2. Pick CG1 and CG2 spins in window 1.
  3. Select the CG2 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  4. Pick the HG2 spin in the orthogonal plane. The correct HG2 spin position should exhibit only CB and CG2 peaks.
  5. Open the new HG2/CG2 strip in window 3. Verify, that HB-HG2, HA-HG2 (possibly H-HG) NOE patterns are present. Adjust the HG2/CG2 positions according to aliphatic 13C-resolved NOESY.
  6. Select the CG1 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  7. Pick HG12 and HG13 spins in the orthogonal plane. The correct HG12 and HG13 spin positions should exhibit CB and CG1 peaks (plus unassigned CD1 peak).
  8. Open the new HG12/CG1 and HG13/CG1 strips in window 3. Verify that HB-HG12, HB-HG13, HG2-HG12, HG2-HG13 (possibly HA-HG12, HA-HG13, H-HG12 and H-HG13) NOE peaks are present. Adjust the HG12/CG1 and HG13/CG1 positions according to aliphatic 13C-resolved NOESY.
  9. Verify HG12 and HG13 assignments in window 5
  10. Select HG12/CG1 or HG13/CG1 strip in window 5.
  11. Pick the CD1 spin in window 1.
  12. Select the CD1 spin and right-click, then Show Orthogonal to display the orthogonal plane.
  13. Pick HD1 spin in the orthogonal plane. The correct HD1 spin position should exhibit CG and CD1 peaks.
  14. Open the new HD1/CD1 strip in window 3. Verify that HB-HD1, HG12-HD1, HG13-HD1, HG2-HD1 (possibly HA-HD1) NOE patterns are present. Adjust the HD1/CD1 position according to aliphatic 13C-resolved NOESY.
  15. Verify assignment by checking all strips in 3D (H)CCH-TOCSY
  16. Validate assignments with 3D 15N-resolved NOESY in window 6. Make sure you observe H-HA, H-HB, H-HG2, possibly H-HG12, H-HG13 and H-HD1 intra peaks, as well as HA(i)-H(i+1) and, possibly, HB(i)-H(i+1), HG12(i)-H(i+1), HG13(i)-H(i+1), HG2(i)-H(i+1), HD1(i)-H(i+1) sequential peaks.

It is exceptionally rare for Ile to have degenerate HG12 and HG13 shifts. On the contrary, they are normally very far apart. Therefore, make sure that you don't miss one of them by zooming in too closely on a spectrum.

HB can overlap with either HG12 or HG13

Filling in the gaps

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

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

-- Main.AlexEletski - 09 Mar 2007

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