NESG Wiki - User contributions [en]

Resonance Assignment/CARA/Backbone assignment GFT

2009-12-08T21:50:31Z

YunfenHe:

=== '''HA And HB Assignment in (4,3)D HABCAB(CO)NHN with CARA''' ===

#Run '''GFT_CreateHABProjSpins''' Lua script to create GFT spins like CApHA-1 and CBpHB-1. Known CA and CB chemical shifts and average HA and HB chemical shifts from assigned residue types will be used. '''IMPORTANT!''' This script will overwrite existing CA+/-HA-1 and CB+/-CB-1 spins!
#Use PolyScope or SynchroScope to move these spins to their appropriate positions. Create these spins if they are not present in a spin system. You can also use StripScope.
#Run '''GFT_HABCAB2HAHBCACB''' Lua script to create HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins. It should also create CA-1 an CB-1 spins if they were missing and report inconsistencies. '''IMPORTANT!''' This script will overwrite existing HA-1 and HB-1 spins!
#Run '''CopyProjectedSpinsToOriginSystem2''' or '''CopyProjectedSpinsToOriginSystem''' Lua script to copy HA-1 and HB-1 to HA and HB spins of successor systems. In CARA jargon "projected spins" refers to spins with non-zero offset.

=== '''HA And HB Assignment in 3D HBHA(CBCACO)NH with CARA''' ===

#Make sure the HBHA(CBCACO)NH is loaded into repository.
#If necessary, adjust the calibration in the H-N plane to match that of 15N-resolved NOESY. Also, adjust the calibration in the Hα/Hβ dimension to match 15N- and 13C-resolved NOESY spectra.
#Proceed with picking HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in assigned fragments. Use HA-1 and HB-1 for spins with degenerate shifts.

*Make sure that the spins are matching the preceding residue type. For example, if the previous residue is Ile, you should pick HB-1, as HB2-1 would be incorrect.
*The standard BioPack pulse sequence <tt>ghbha_co_nh.c</tt> employs 1H multiplicity editing. Thus, cross-peaks of CH2 groups will have the opposite sign that of CH and CH3 (Ala HB). The advantage of this is the additional information on amino acid typing. The drawbacks are possible mutual signal cancellations, within Ser and Thr spin systems, or between spin systems, which overlap in the H-N plane. 

#Create empty spin systems for each unassigned residue preceding an assigned fragment. This is, typically, the case with prolines. Link each empty system to adjacent assigned fragments. 
#Run '''CopyProjectedSpinsToOriginSystem''' Lua script to copy "projected spins" (with offset -1) to preceding spin systems. You have to run it 6 times, for HA-1, HA2-1, HA-3, HB-1, HB2-1 and HB3-1.

*Empty spin systems will thus become populated - this is the reason for creating them in the previous step.
*Existing HA, HA2, HA3, HB, HB2 and HB3 spins (if any) will be updated with new chemical shifts. You may want to preserve this information by saving the repository before running '''CopyProjectedSpinsToOriginSystem'''.

== Additional backbone assignment and verification ==
# Verify the backbone assignment by tracing HA(i) <-> HN(i+1) and HB(i) <-> HN(i+1) connectivities in the 15N-resolved NOESY spectrum.
# Pick HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in unassigned systems, if your backbone assignment is incomplete.
# Try to complete the backbone assignment by matching 15N-resolved NOESY strips. If reliable assignment cannot be established, postpone completion until the side-chain assignment is complete.

%COMMENT%

-- Main.AlexEletski - 06 Jul 2007

Resonance Assignment/CARA/Backbone assignment GFT

2009-12-08T21:50:06Z

YunfenHe:

Resonance Assignment/CARA/Backbone assignment GFT

2009-12-08T21:38:56Z

YunfenHe:

=== '''HA And HB Assignment in (4,3)D HABCAB(CO)NHN with CARA''' ===

#Run '''GFT_CreateHABProjSpins''' Lua script to create GFT spins like CApHA-1 and CBpHB-1. Known CA and CB chemical shifts and average HA and HB chemical shifts from assigned residue types will be used. '''IMPORTANT!''' This script will overwrite existing CA+/-HA-1 and CB+/-CB-1 spins!
#Use PolyScope or SynchroScope to move these spins to their appropriate positions. Create these spins if they are not present in a spin system. You can also use StripScope.
#Run '''GFT_HABCAB2HAHBCACB''' Lua script to create HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins. It should also create CA-1 an CB-1 spins if they were missing and report inconsistencies. '''IMPORTANT!''' This script will overwrite existing HA-1 and HB-1 spins!
#Run '''CopyProjectedSpinsToOriginSystem2''' or '''CopyProjectedSpinsToOriginSystem''' Lua script to copy HA-1 and HB-1 to HA and HB spins of successor systems. In CARA jargon "projected spins" refers to spins with non-zero offset.

=== '''HA And HB Assignment in 3D HBHA(CBCACO)NH with CARA''' ===

# Make sure the HBHA(CBCACO)NH is loaded into repository.
# If necessary, adjust the calibration in the H-N plane to match that of 15N-resolved NOESY. Also, adjust the calibration in the Hα/Hβ dimension to match 15N- and 13C-resolved NOESY spectra.
# Proceed with picking HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in assigned fragments. Use HA-1 and HB-1 for spins with degenerate shifts.
** Make sure that the spins are matching the preceding residue type. For example, if the previous residue is Ile, you should pick HB-1, as HB2-1 would be incorrect.
** The standard BioPack pulse sequence <tt>ghbha_co_nh.c</tt> employs 1H multiplicity editing. Thus, cross-peaks of CH2 groups will have the opposite sign that of CH and CH3 (Ala HB). The advantage of this is the additional information on amino acid typing. The drawbacks are possible mutual signal cancellations, within Ser and Thr spin systems, or between spin systems, which overlap in the H-N plane.
# Create empty spin systems for each unassigned residue preceding an assigned fragment. This is, typically, the case with prolines. Link each empty system to adjacent assigned fragments.
# Run '''CopyProjectedSpinsToOriginSystem''' Lua script to copy "projected spins" (with offset -1) to preceding spin systems. You have to run it 6 times, for HA-1, HA2-1, HA-3, HB-1, HB2-1 and HB3-1.
** Empty spin systems will thus become populated - this is the reason for creating them in the previous step.
** Existing HA, HA2, HA3, HB, HB2 and HB3 spins (if any) will be updated with new chemical shifts. You may want to preserve this information by saving the repository before running '''CopyProjectedSpinsToOriginSystem'''.

== Additional backbone assignment and verification ==
# Verify the backbone assignment by tracing HA(i) <-> HN(i+1) and HB(i) <-> HN(i+1) connectivities in the 15N-resolved NOESY spectrum.
# Pick HA-1 (or HA2-1 and HA3-1) and HB-1 (or HB2-1 and HB3-1) spins in unassigned systems, if your backbone assignment is incomplete.
# Try to complete the backbone assignment by matching 15N-resolved NOESY strips. If reliable assignment cannot be established, postpone completion until the side-chain assignment is complete.

%COMMENT%

-- Main.AlexEletski - 06 Jul 2007

CARA vs Xeasy

2009-12-08T20:49:43Z

YunfenHe:

Below is concise list of key features of CARA as compared to [[XEASY]] that should help new users make a transition.

==== Data Organization ====

[[XEASY]] keeps data stored in separate files: spectra, sequence lists, atom lists, and peaklists.

CARA stores all data in a "repository" - an XML file, which contains all information (residue type definitions, spectrum types definitions, spin systems, spins, peak lists, extension scripts, paths to NMR spectra, etc.). This file typically has a .cara extension. A repository is built from a template by adding "projects". This approach allows much better control and overview over a structure project and makes sharing data among researchers easier.

Though CARA protects repositories from modifications, which may compromise its stability, repositories can be edited with a plain text or XML editor.

==== Atom Nomenclature ====

Current repository templates are designed to be compatible with the BMRB format. That is, H is used instead of HN, and glycines have HA2/3 instead of HA1/2. This is also the default format of '''cyana.lib '''in [[CYANA]] 2.1.

In addition to that, pseudoatoms are labeled as H* instead of Q* and QQ*. For example, HB is used for QB, HD1/2 for QD1/2, and HD for QQD of Leu. This is compatible with the '''pseudo=2''' setting in [[CYANA]] 2.1

==== Residues and Spin Systems ====

XEASY stores information about spin systems (also referred to as fragments in XEASY manual) and residues in the same *.seq file, and requires somewhat confusing swapping of mapping and fragment numbers for sequence-specific assignment.

CARA clearly distinguishes residues and spin systems as separate classes in repository database. Spin systems can be numbered from 1 upward. They can be linked into fragments by setting successor and predecessor tags. Fragments can be "mapped" to the sequence to see if they match a particular stretch of residues. A spin system becomes assigned when its assignment tag is set, pointing to a certain residue in the sequence.

==== Atoms and Spins ====

What XEASY refers to as atoms are called "spins" in CARA. "Atoms" in CARA form a different data class - it is a member of "residue type" as a part of molecular structure. The database of spins can be exported as an XEASY atom list, and an XEASY atom list can be imported into CARA (provided that the nomenclature matches).

While you may need to use separate atom lists with different spectra and peak lists in XEASY, CARA works with a single set of spins. Spin aliases are used in situations where the same spin has different chemical shifts (see below).

==== Spin Labels ====

Spin labels have the form [?|!]SPINLABEL[+|-x], where SPINLABEL is an alphanumeric string and +x or -x is the "offset". The question mark ? as the first character marks temporary assignments, the exclamation sign "!" - stereospecific assignments. Since + and - characters are reserved to specify offsets, lowercase letters "p" and "m" should be used instead for spin labels in GFT spectra. For example, sequential CA+CB peak should be labeled CApCB-1.

Offset designations in CARA carry more information than in XEASY - they are used to search for sequential neighbors.

==== Peak Inference and Peak Lists ====

Though peak lists in XEASY format can be exported from any spectrum, they only need to be generated for NOESY spectra to provide input to programs like CYANA and AutoStructure. They are not needed for backbone and side-chain assignment.

Peak marks, which you see in spectrum display tools ( "scopes", in this case other than MonoScope ) are displayed at positions, inferred from chemical shifts, and residue type and spectrum type definitions. Spectrum type definitions describe the correlations prouced in a given spectrum and valid spin labels in every dimension. This is equivalent to XEASY peaklists being generated from an atom list on-the-fly. If a peak is moved in CARA, then the chemical shifts of the spins involved are updated instantly. If a new peak is picked, new spins are created. These changes are synchronized across all "scopes".

==== Spectral Folding and Aliasing ====

In CARA all spins and peaks have their exact assignments and positions (in ppm). There are no folding attributes for peaks, as in XEASY. Instead, it is possible to navigate beyond the spectrum edge to display the true unfolded location.

==== Spin Links ====

Spin links do not have a direct equivalent in XEASY. They are designed to describe correlations observed in NOESY spectra, but CARA does not prescribed a unique use for them. Spin links present a good means of visualizing UPLs or short distances in NOESY spectra. Spin links can also be used instead of peak list in manual NOE assignment.

A useful feature is that a single spin link produces two peaks - the direct and transposed. Spin links and inferred peaks can be exported as XEASY peak lists.

==== Spin Aliases ====

There may be significant mismatches between related NMR spectra. Typical causes are different sample and experimental conditions, such as temperature differences or non-resonant effects, or systematic offsets, such as those between TROSY and conventional NMR spectra. In such cases spin aliases can be helpful.

The spin alias object is a child of spin. It has its own chemical shift (typically different from that of spin itself), and a tag with the ID of the spectrum, where this alias was set. Spin aliases have a purely cosmetic effect on spectral appearance, defining positions, where planes and and slices will be taken.

It is recommended to have all spectra calibrated and matched as best as possible, before resorting to spin aliases. Also you would want to have the main chemical shifts set to match the NOESY spectra, since they are exported for use in automated structure calculation, and use aliases for other spectra, such as HCCH-COSY.

==== Scripting ====

CARA functionality is extended by scripts in Lua programming language ([http://www.lua.org/ www.lua.org]). Besides default Lua functionality interface to many CARA functions is available.

-- AlexEletski - 06 Mar 2007

[[Category:CARA]][[Category:Resonance_Assignment]][[Category:XEasy]]

CARA vs Xeasy

2009-12-08T20:45:00Z

YunfenHe:

Below is concise list of key features of CARA as compared to [[XEASY]] that should help new users make a transition.

==== Data Organization ====

[[XEASY]] keeps data stored in separate files: spectra, sequence lists, atom lists, and peaklists.

CARA stores all data in a "repository" - an XML file, which contains all information (residue type definitions, spectrum types definitions, spin systems, spins, peak lists, extension scripts, paths to NMR spectra, etc.). This file typically has a .cara extension. A repository is built from a template by adding "projects". This approach allows much better control and overview over a structure project and makes sharing data among researchers easier.

Though CARA protects repositories from modifications, which may compromise its stability, repositories can be edited with a plain text or XML editor.

==== Atom Nomenclature ====

Current repository templates are designed to be compatible with the BMRB format. That is, H is used instead of HN, and glycines have HA2/3 instead of HA1/2. This is also the default format of '''cyana.lib '''in [[CYANA]] 2.1.

In addition to that, pseudoatoms are labeled as H* instead of Q* and QQ*. For example, HB is used for QB, HD1/2 for QD1/2, and HD for QQD of Leu. This is compatible with the '''pseudo=2''' setting in [[CYANA]] 2.1

==== Residues and Spin Systems ====

XEASY stores information about spin systems (also referred to as fragments in XEASY manual) and residues in the same *.seq file, and requires somewhat confusing swapping of mapping and fragment numbers for sequence-specific assignment.

CARA clearly distinguishes residues and spin systems as separate classes in repository database. Spin systems can be numbered from 1 upward. They can be linked into fragments by setting successor and predecessor tags. Fragments can be "mapped" to the sequence to see if they match a particular stretch of residues. A spin system becomes assigned when its assignment tag is set, pointing to a certain residue in the sequence.

==== Atoms and Spins ====

What XEASY refers to as atoms are called "spins" in CARA. "Atoms" in CARA form a different data class - it is a member of "residue type" as a part of molecular structure. The database of spins can be exported as an XEASY atom list, and an XEASY atom list can be imported into CARA (provided that the nomenclature matches).

While you may need to use separate atom lists with different spectra and peak lists in XEASY, CARA works with a single set of spins. Spin aliases are used in situations where the same spin has different chemical shifts (see below).

==== Spin Labels ====

Spin labels have the form [?|!]SPINLABEL[+|-x], where SPINLABEL is an alphanumeric string and +x or -x is the "offset". The question mark ? as the first character marks temporary assignments, the exclamation sign "!" - stereospecific assignments. Since + and - characters are reserved to specify offsets, lowercase letters "p" and "m" should be used instead for spin labels in GFT spectra. For example, sequential CA+CB peak should be labeled CApCB-1.

Offset designations in CARA carry more information than in XEASY - they are used to search for sequential neighbors.

==== Peak Inference and Peak Lists ====

Though peak lists in XEASY format can be exported from any spectrum, they only need to be generated for NOESY spectra to provide input to programs like CYANA and AutoStructure. They are not needed for backbone and side-chain assignment.

Peak marks, which you see in spectrum display tools ( "scopes", in this case other than MonoScope ) are displayed at positions, inferred from chemical shifts, and residue type and spectrum type definitions. Spectrum type definitions describe the correlations prouced in a given spectrum and valid spin labels in every dimension. This is equivalent to XEASY peaklists being generated from an atom list on-the-fly. If a peak is moved or in CARA, then the chemical shifts of the spins involved are updated instantly. If a new peak is picked, new spins are created. These changes are synchronized across all "scopes".

==== Spectral Folding and Aliasing ====

In CARA all spins and peaks have their exact assignments and positions (in ppm). There are no folding attributes for peaks, as in XEASY. Instead, it is possible to navigate beyond the spectrum edge to display the true unfolded location.

==== Spin Links ====

Spin links do not have a direct equivalent in XEASY. They are designed to describe correlations observed in NOESY spectra, but CARA does not prescribed a unique use for them. Spin links present a good means of visualizing UPLs or short distances in NOESY spectra. Spin links can also be used instead of peak list in manual NOE assignment.

A useful feature is that a single spin link produces two peaks - the direct and transposed. Spin links and inferred peaks can be exported as XEASY peak lists.

==== Spin Aliases ====

There may be significant mismatches between related NMR spectra. Typical causes are different sample and experimental conditions, such as temperature differences or non-resonant effects, or systematic offsets, such as those between TROSY and conventional NMR spectra. In such cases spin aliases can be helpful.

The spin alias object is a child of spin. It has its own chemical shift (typically different from that of spin itself), and a tag with the ID of the spectrum, where this alias was set. Spin aliases have a purely cosmetic effect on spectral appearance, defining positions, where planes and and slices will be taken.

It is recommended to have all spectra calibrated and matched as best as possible, before resorting to spin aliases. Also you would want to have the main chemical shifts set to match the NOESY spectra, since they are exported for use in automated structure calculation, and use aliases for other spectra, such as HCCH-COSY.

==== Scripting ====

CARA functionality is extended by scripts in Lua programming language ([http://www.lua.org/ www.lua.org]). Besides default Lua functionality interface to many CARA functions is available.

-- AlexEletski - 06 Mar 2007

[[Category:CARA]][[Category:Resonance_Assignment]][[Category:XEasy]]

Resonance Assignment/CARA

2009-12-08T20:33:26Z

YunfenHe:

== Loading a new template ==

To start a new structure determination project in CARA you need to load a template. A template is a CARA repository without project that contains definitions for residue types, spectrum type and LUA scripts. You can either load the default template from the [http://www.cara.ethz.ch/Wiki/TemplatesPage templates page] of the official CARA web-site, or extract a template from an existing CARA repository.

In CARA click '''File -> New from template''' and select the appropriate <tt>.cara</tt> file. For detailed instructions see [http://www.cara.ethz.ch/Wiki/ImportingTemplate]

 [[Image:CARA New Template Dialogue.png]]

CARA Template for GFT spectra [[Media:BoR54_template.cara|BoR54_template.cara]]: Latest GFT template for BoR54 project

== Starting a new project ==

Normally you would create a new project based on a protein sequence in XEASY format.

If you have the protein sequence in one-letter format you can convert it to XEASY <tt>.seq</tt> file the following way:
#Save the one-letter sequence in a <tt>.aa</tt> file, e.g. <tt>foo.aa</tt>
#Execute '''OneLetterFileToSeqFile''' LUA script from '''Terminal''' tab of the main window. Select your <tt>.aa</tt> file as input and give full path for the output <tt>.seq</tt> file.

[[Image:CARA One Letter to Seqfile.png]]

To create a new project for you protein in the repository right-lick '''Projects''' -> '''Import Project from Sequence...'''. Select the corresponding <tt>.seq</tt> file in the file explorer and click on the '''Residues''' button in the pop-up menu.

[[Image:CARA proj import from seq.png]]

This will create a project entry with the same name as the *.seq file, with the sequence field appropriately filled and all other fields empty. The new project will have the same name as the provided <tt>.seq</tt> file, though it can be renamed.

[[Image:CARA new project.png]]

Even though a repository can contain several projects, it is a good practice to have only one project per repository. For example, many LUA scripts assume that there is a single project in a repository.

You can also import a project from a BMRB file. In this case chemical shift data will be imported in addition to the sequence. For more details see [http://www.cara.ethz.ch/Wiki/CreateNewProject]. Also check the page on importing XEASY files: [http://www.cara.ethz.ch/Wiki/WorkingWithOtherProgramsImportExport]

== Importing NMR spectra ==

The next step is to import NMR spectra into the project. Left-click on the '''Spectra''' node in the project, then right-click in the panel to the right. In the context menu move the mouse cursor to '''Add Spectrum''' and select the appropriate spectrum type, then chose the spectrum file in the file explorer. The following formats can be imported: XEASY (<tt>.3D.param</tt>), Sparky (<tt>.ucsf</tt>), BRUKER (<tt>.rr</tt>), NMRPipe (<tt>.ft</tt>) and Felix (<tt>.mat</tt>).

Most common spectra types are defined in the standard template. Please note that in the default template there are distinct spectrum type definitions for aliphatic and aromatic versions 2D [13C,1H] HSQC, 3D 13C-resolved NOESY, and HCCH spectra. You can also define additional spectrum types as described in [http://www.cara.ethz.ch/Wiki/CreateSpectrumType http://www.cara.ethz.ch/Wiki/CreateSpectrumType].

The order of spectral dimensions is normally irrelevant as CARA determines the correct mapping to the spectrum type from the type of nucleus associated each dimension. However, if the mapping is not unique (as in the case of 3D HBHA(CO)NH, 3D heteronuclear-resolved NOESY, 3D (H)CCH, 3D H(C)CH spectra, etc.), then a pop-up widow will prompt you to define the correct mapping.

[[File:CARA_map_to_spectrum_type.png]]

For a given dimension CARA can "guess" the corresponding nucleus type from the ppm range of the dimension. This, however, may still be insufficient to distinguish between 15N and carbonyl 13C dimensions, or between methyl 13C and 1H. For XEASY spectra one solution to this problem is to make sure before importing that <tt>.3D.param</tt> include <tt>Identifier for dimension wX</tt> lines as in this example for a 3D HNCO spectrum:
<pre>
Version ....................... 1
Number of dimensions .......... 3
16 or 8 bit file type ......... 16
Spectrometer frequency in w1 .. 60.7413
Spectrometer frequency in w2 .. 150.7300
Spectrometer frequency in w3 .. 599.4460
Spectral sweep width in w1 .... 27.6576
Spectral sweep width in w2 .... 15.9225
Spectral sweep width in w3 .... 8.3410
Maximum chemical shift in w1 .. 131.7220
Maximum chemical shift in w2 .. 185.6120
Maximum chemical shift in w3 .. 13.1120
Size of spectrum in w1 ........ 128
Size of spectrum in w2 ........ 256
Size of spectrum in w3 ........ 512
Submatrix size in w1 .......... 16
Submatrix size in w2 .......... 32
Submatrix size in w3 .......... 64
Permutation for w1 ............ 3
Permutation for w2 ............ 2
Permutation for w3 ............ 1
Folding in w1 ................. RSH
Folding in w2 ................. RSH
Folding in w3 ................. RSH
Type of spectrum .............. ?
Identifier for dimension w1 ... N
Identifier for dimension w2 ... C
Identifier for dimension w3 ... H
</pre>

For more details see [http://www.cara.ethz.ch/Wiki/ImportSpectra]

== Referencing and aliasing in NMR spectra ==

There could be small systematic shifts even between properly transformed spectra. An easy way to correct these offsets interactively is described in the instructions for using SynchroScope [http://www.cara.ethz.ch/Wiki/SynchroScope]. Another possibility is to right-click on the spectrum of interest in the main window and select '''Calibrate Spectrum...''' from the context menu. You can explicitly enter the required corrections (in ppm) in in the pop-up window.

Please note that these are corrections for systematic shifts only - you cannot fix incorrect spectral widths within CARA. These calibration corrections are internal to CARA and the original XEASY <tt>.3D.param</tt> files remain unchanged. To properly use the same spectra and CARA assignments with an external program, such as ATNOSCANDID, you have to export the CARA calibration by right-clicking on the spectrum tab and selecting '''Write Calibration...''' from the context menu, which will update the corresponding <tt>.3D.param</tt> files.

In contrast to XEASY, peaks and spins in CARA are always shown at their true chemical shifts and lack folding attributes. To see folded peaks you have to scroll the spectrum beyond its default range in any given display scope (the '''View''' -> '''Show Folded''' checkbox must be selected to display folded regions, otherwise they will be black).

Folding attributes of spectral dimensions determine how "unfolded" regions are generated. CARA interprets the dimension folding attributes <tt>Folding in wX</tt> of <tt>.3D.param</tt> files upon importing, which have to be either RSH or TPPI. RSH stands for the most common wrap-around aliasing and TPPI means mirror-reflection folding. You can also change folding attributes after a spectrum is imported. Just expand the spectrum node to see the dimensions, right-click on a dimension tab and select '''Set Folding...''' from the context menu.

For additional details and examples see [http://www.cara.ethz.ch/Wiki/FoldingAndAliasing] .

 

 

#[[CARA Introduction|Introduction]]
#[[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]]

Resonance Assignment/Principles and concepts

2009-12-08T19:12:54Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

*

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide.
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 

    The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.

*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence-specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionally, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggest the best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin systems linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and then uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T19:08:36Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

*

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide.
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 

    The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.

*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence-specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionally, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggest the best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T18:56:52Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

*

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide.
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 

    The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.

*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence-specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionally, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggestthe best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T18:52:15Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

*

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide.
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 

    The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.

*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence-specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionaly, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggestthe best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T18:47:21Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.

*

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 
**The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.
*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionaly, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggestthe best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T18:46:40Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shift. Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has.
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain.
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues.
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs.
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO, it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C.
*All HCCH-type spectra can also be tailored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.


 

 

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 
**The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.
*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionaly, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggestthe best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.

Resonance Assignment/Principles and concepts

2009-12-08T18:23:08Z

YunfenHe:

== Introduction ==

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see ''e.g.'' Kurt Wütrich's book ''NMR of Proteins and Nucleic Acids'' (Wiley, 1986) and John Cavangh's ''et al''. textbook ''Protein NMR Spectroscopy. Principles and Practice'' (2nd edition, Elsevier, 2007). 

== Stable isotope labeling schemes ==

 

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows: 

*100% 15N, 100%13C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation. 
*100% 15N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data. 
*100% 15N, 5-7% 13C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
*100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination. 

 

In addition to these labeling schemes, one can find it is useful, especially for larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level. 

== NMR experiments ==

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site. 

=== Through bond ===

Through-bond experiments correlate nuclei connected by a limited number of chemical bonds.

*15N-HSQC - correlates a 15N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups. 
*13C-HSQC - correlates a 13C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of 13C chemical shifts 
**Constant time version enhances resolution. Peak sign gives information on the number of 13C neighbors a given 13C nucleus has
*HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a Cγ/Cδ of Asn/Gln with NH2 of the side chain
*HNCA, 3D - correlates a backbone amide with the Cα of the same and preceding residues
*HNCACB, 3D - correlates a backbone amide with the Cα and Cβ of the same and preceding residues. Usually has Cβ and Cα peaks of opposite signs
*CBCA(CO)NH, 3D - correlates a backbone amide with the Cα and Cβ of the preceding residue. Together with HNCACB, HNCA and HNCO it is used to assign the backbone chemical shifts. 
*HBHA(CBCACO)NH, 3D - correlates a backbone amide with the Hα and Hβ resonances of the preceding residue. Used together with HCCH-type experiments and 13C-HSQC to assign the side chain resonances. 
*H(C)CH-COSY, 3D - correlates an aliphatic C-H pair with an adjacent aliphatic 1H of the same side chain. Closely related experiment is H(C)CH-TOCSY, 3D, which correlates an aliphatic C-H pair with all aliphatic 1H nuclei of the same side chain.
*(H)CCH-COSY, 3D, and (H)CCH-TOCSY, 3D, are very similar to above, except the third correlated nucleus is 13C
*All HCCH-type spectra can also be taylored for the aromatic region of 13C range. Correlations would be detected only within an individual aromatic ring.
*(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE, 2D - correlate Hδ and Hε aromatic protons with Cβ of the same residue. Useful for assigning chemical shifts of nuclei in aromatic rings.


 

*Long range 15N-HSQC, 2D - correlates Hδ2 and Hε1 protons (attached to 12C or 13C nuclei!) on His rings with Nδ1 and Nε2 nuclei. This experiment s useful for assigning Hε1 and Cε1 nuclei of His rings as well as to determine the protonation state of the ring.

=== Through space ===

Through-space experiments, as the name implies, correlate pairs of nuclei close in space. Many such nuclei would belong to the same or adjacent residues and these experiments can thus supplement the information obtained from through-bond spectra. The usual distance limit to observe such a correlation is ~5Â.

*15N-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of an amide group with both nuclei of the amide
*13C-edited NOESY-HSQC, 3D - correlates all protons close to the 1H of a C-H moiety with both nuclei of the moiety. Can replace an H(C)CH-TOCSY experiment in certain cases. 
**The above spectrum can be separated into aliphatic and aromatic regions of the 13C range.
*Simultaneous 13C,15N-edited NOESY-HSQC, 3D - as the name implies, this spectrum combines the above two experiments (15N and 13C).

These NOESY-type spectra are very useful in confirming the sequence specific backbone assignments and in connecting aromatic rings to their proper side chains. For certain automatic methods (e.g. [[Abacus|ABACUS]]) they contain all necessary sequencial information and can thus completely replace HNCACB or similar spectra.

== Spin systems ==
[[Image:PBfragment.jpg|thumb|400px]]
=== Definition  ===

A spin system in the broadest sense of the word is a set of nuclei connected by chemical bonds. While for small molecules this is a very useful definition, for proteins it becomes unwieldy as the whole protein would thus be just one spin system. In practice, in protein NMR, a '''spin system''' is usually defined as an amino acid residue (AA-fragment) - N, HN, Cα, Hα, C', and all nuclei of the side chain. 

Sometimes it may be more convenient to define a '''peptide-bond fragment''' (PB-fragment) spin system, which would contain N and HN of the following amino acid, instead of the same. See figure (left). 

Occasionaly, an aromatic ring may be treated as a separate spin system from the rest of the same side chain.

=== [[Image:SpinSystemID.jpg|thumb|left|400px]]Identification ===

Whether one uses AA- or PB-fragments (see above), a convenient handle on the spin system is the backbone amide group, as there is only one per each residue. Prolines would have no such handle and are treated separately.

One thus normally starts the resonance assignment project with picking the 15N-HSQC spectrum and assigning each peak a unique identifier (Figure, right), which is later used as an identifier of the whole spin system.

=== Linking spin systems ===

Once identified, the spin systems can then be linked using information from relevant pairs of spectra, such as HNCACB and CBCA(CO)NH, HN(CO)CA and HNCA, HNCO and HN(CA)CO. Each software package has a slightly different method to do that, but generally for manual linking one looks for identical 13C chemical shifts in intraresidual and sequential spectra, simultaenously displaying strips from the above pairs. The programs often suggestthe best fit. Some programs have built-in optimization functions. 

Many automatic programs (MONTE, MARS, [[The PINE Server|PINE]], [[AutoAssign|AutoAssign]]) only require peak lists from the above spectra (and 15N-HSQC) and use sofisticated algorithms to link the spin systems sequentially and match them onto the protein sequence.

=== Matching onto covalent structure ===

Some amino acids have very characteristic Cα and Cβ chemical shifts (e.g. Ala, Gly, Ser, Thr) while others can be easily mixed up (Phe/Tyr/Asp/Asn/Cys or Gln/Glu/Met). However, once a few spin systems form a short chain, one can start looking for a proper match with the protein sequence. Again, most software package would suggest best fits. 

After the initial match, spin system linking and sequential backbone assignment often go hand in hand. Once this step is concluded, normally one includes the rest of the side chain nuclei through HCCH and NOESY-type spectra, after which one adds aromatic rings, side chain NH2 groups of Asn and Gln, methyl groups of Met, etc.

Unlike the above process, [[Abacus|ABACUS]] software first expects individual spin systems be as complete as possible, with side chain information included, and the uses NOESY-type spectra to complete the assignment via Monte Carlo simulations.