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Hcch Tocsy Assignment Submission

References:
A. Bax, G.M. Clore and A.M. Gronenborn (1990) J. Magn. Reson.88 425-431. (Link to Article)
E.T. Olejniczak, R.X. Xu and S.W. Fesik (1992) J. Biomol. NMR2 655-659. (Link to Article)

Minimum labelling:15N, 13C

Dimensions: 3

Magnetisation is transferred from the side-chain hydrogen nuclei to their attached 13C nuclei. This is followed by isotropic 13C mixing and finally transfer back to the side-chain hydrogen atoms for detection.

This spectrum is used for side-chain assignment. It yields strips at each carbon frequency in the side chain in which all side-chain hydrogen resonances are visible. The HCCH-COSY is a slightly less crowded version in which only the hydrogen resonances of the own and neighbouring carbons are visible. Note that the spectrum is usually displayed in such a way that the 1H detected dimension is shown along the y-axis rather than the x-axis. This means that the peaks belonging to one side-chain appear in a vertical strip rather than a horizontal strip.

Experiments : Experiments¶

Curate Experiment Parameters

This popup window is used to control parameters that relate to the NMR experiment entities within a CCPN project. It should be noted that an experiment is specifically a record of what was done, not a reference to the data that was obtained; this is what Analysis refers to as a spectrum. Also, an experiment doesn’t just refer to what kind of NMR experiment was performed, but rather to something that was done on a particular occasion to a particular sample. The kinds of information that are stored at the experiment level describe how the experiment was performed and how certain data that derives from the experiment should be interpreted. Several tabs are used to sub-divide the popup window into several sections to control different aspects of the experiments.

Main “Experiments” Tab

This table is the main display of all if the NMR experiments described within the CCPN project. Each experiment may refer to one or more spectra (and even an FID) that resulted from the experimental operation; the names of these are listed in the “Data Sources” column. Several experimental parameters may be adjusted in this table and, as far as resonance assignment is concerned, the most important of these are the “Shift List” and “Mol Systems”. It should be noted that the “Shift Ref” column can only be configured if chemical shift reference details have been entered at the “Shift References” tab.

The shift list of an experiment states how chemical shift measurements should be grouped. An experiment’s assignments, on the peak of its spectra, only contribute to the chemical shift measurenemts in one shift list. In normal operation each shift list corresponds to a given set of conditions, where resonance positions in spectra a fairly static. Different experiments may use different shift lists if their sample conditions are different enough to cause peaks to move. Accordingly, a resonance derived from a given atom may have several different recorded chemical shift values, each of which resides in a different chemical shift list. Because each experiment is associated with a single shift list it is thus known which chemical shift average the assignments in its spectra contribute to and which chemical shift values to look at when suggesting assignments for peaks. The shift list that an experiment is linked to may be changed at any time, and when an experiment is moved from one shift list to another (which may be new and empty) the contributions that its spectrum peaks make to the calculation of average chemical shift values will automatically be adjusted; the experiments that are linked to a given shift list dictate which peaks to average chemical shift values over.

The “Mol Systems” for an experiment specify which molecular systems were present in the sample. In essence this means which group of molecular chains the spectrum peaks, that come from the experiment, could be assigned to (caused by). In normal operation the molecular information need not specifically be set in this table, because the connection is made automatically when a peak is assigned to a resonance that is known to be from a specific atom in a specific residue. Once an experiment is associated with a particular molecular system, subsequent attempts to assign its peaks to atoms in a different molecular system will trigger a warning. The molecular system for an experiment may nonetheless be set via this table. Sometimes this is to preemptively associate particular experiments, and hence spectra, with specific molecular systems so that there is less chance of accidentally assigning a peak to the wrong thing. The molecular system, and hence residues, that an experiment is linked to is used to facilitate several operations in Analysis. For example, when operating on a peak to associate one of its assigned resonances with an atom, the molecular system link allows the correct group of chains to be displayed automatically.

Experiment Types

This table is used to specify what kind of NMR experiment was done for each of the experiment records within the project. The general idea is that the basic magnetisation transfer pathway is given and how this relates to the experimental dimensions. Setting such information helps facilitate several operations in Analysis. For example when making NMR derived distance restraints only peaks from “through-space” experiments like NOESY are listed. Also, and perhaps most importantly, the linking of experimental dimensions to references, which correspond to particular points of the magnetisation transfer pathway, enables the assignment system to have knowledge of experimental dimensions that are linked together via covalent “one-bond” connectivity. Thus, if an experiment is set to be of 15N HSQC type, then it is known that any spectrum peaks represent one-bond correlations between hydrogen and nitrogen. This dictates what the isotope type of any assignments must be, and if a peak dimension is assigned to a specific atom then the assignment on the other peak dimension must be to the covalently bound atom.

In normal operation the user specifies the type of NMR experiment that was run by selecting options in the “Category”, “Type Synonym” and “Full Type” columns. If the full CCPN name of the experiment type is known then the user can go straight for the “Full Type”, but selecting the category and/or synonym first allows the number available options to be reduced dramatically; without this all possible experiment types that have matching isotopes are shown. Setting the category for the NMR experiment gives a sub division between through-bond, through-space, quantification and other types. Strictly speaking an experiment may belong to more than one category, but in this system it is only listed in the least populous. For example a 15N HSQC-NOESY has both through-bond and though-space transfers but is categorised as through-space. If the category for an experiment is unknown, or not particularly helpful, the user may set the synonym in the first instance. The “synonym” of an experimental type in Analysis is a common human-readable name, for example “HNCA” or “15N HSQC NOESY”, but this may still not be sufficient to fully specify the exact NMR experiment that was run. To do this the full CCPN type should be considered. The External Source and corresponding name columns are only used in situations where the loading of a spectrum specifies what kind of experiment was run. At present this only occurs for data loaded from Bruker files, and then only if the pulse sequence name in the parameters is known to the system. Nevertheless, if this data is present the experiment type information can be automatically be filled in.

The full CCPN type for an experiment uses a special nomenclature that is described in the CCPN experiment nomenclature paper (slightly out of date now). In essence the user can distinguish between different magnetisation transfer pathways, some of which may have the same common name (synonym). For example a 15N HSQC-TOCSY could have either the HSQC step or the TOCSY step first. In this instance the system offers a choice between H183_H.TOCSY (HSQC first) and H_H[H].TOCSY (TOCSY first). The experiment naming system for the full CCPN type is fairly complex, and is designed to give a precise specification of the magnetisation steps, which atom sites they visit and what measurements are made; giving rise to an experimental dimension. It should be noted however, that this system does not describe the precise NMR pulse sequence that was used. For example no distinction is made between HSQC and HMQC. The essential features of the nomenclature are as follows: capital letters indicate atom sites that were recorded and result in an experimental dimension; lower case letters are atom sites that are part of the pathway but not recorded, e.g. carbonyl in H[N[co[CA]]]; square brackets represent out-and-back transfers; curly brackets with “|” sub-divisions represent alternative pathways; underscores represent transfers that are not one-bond (the transfer type is listed after at the end after a dot).

The lower tables are used to show how the experimental dimensions relate to the reference dimensions that are described by the experiment type database. Sometimes when an experiment type is set it will not be possible to automatically determine which of the experimental dimensions relates to which part in the magnetisation transfer pathway. For example a 3D HCCH TOCSY experiment (full type HC_cH.TOCSY) has two hydrogen dimensions, but only one of these has a one-bond relationship to the carbon dimension. Getting this setting right is crucial for the correct assignment and interpretation of spectra. Sometimes Analysis guesses wrong and the user has to set the correct dimension mapping, thus checks should be made for experiments that have two dimensions with the same kind of isotope. Getting the dimension mapping correct is a matter of looking in the lower left table and seeing how one dimension relates to another. Each dimension that has transfer to another recorded dimension is listed. For example, in an HCCH TOCSY dimension 1 (hydrogen) might be ‘onebond’ to dimension 3 (carbon), but the user may know that it is actually dimension 2 that is really ‘onebond’ to the carbon. This problem may be fixed by double-clicking in the “Transfers To Dim” to swap the hydrogen dimensions; so that dimension 3 (carbon) is listed as ‘onebond’ to dimension 2 (hydrogen). Here, the order of dimensions (and hence number) is the same as that presented when assigning a peak, i.e. in the Assignment Panel.

The lower right “Reference Dimension Mapping” is an alternative way of looking at the same information and shows how the experimental dimensions have been mapped to their reference counterpart in the experiment type database. Here, the “Ref Measurement” column can be used to follow the steps in the magnetisation transfer pathway by following increasing measurement numbers. Changing the “Ref Exp Dim” column in this table is equivalent to making changes in the lower left table, but is perhaps more difficult to understand.

Experimental Details, Instruments and Shift References

The “Experimental Details” table is used to list and edit details about the recorded experiments in terms of its physical setup. The user may specify which instruments were used and information about the sample and NMR tube. It should be noted that in order to specify a spectrometer or probe the specification for the instrument must first be entered in the “NMR Instruments” tab. Currently, none of the NMR details given in this table have any influence on resonance assignment or NMR data analysis, although spinning information may be used for solid-state spectra at some point. However, any experimental details entered into the CCPN project will be present when submissions to the BioMagResBank database are made; initially using the CcpNmr ECI.

The “Shift References” table is use to enter chemical shift reference information into the CCPN project. This may them be linked to experiments via the first “Experiments” tab, and such information is required for database deposition. To add a chemical shift reference specification the user first clicks on either “Add Internal Reference” (internal to the sample) or “Add External Reference” as appropriate. Then for the rows that appear in the table the user double-clicks to edit the columns to specify: which atom in which kind of molecule was used, what the reference value and unit is, and whether the reference is direct or indirect. A reference atom allows the direct referencing of resonances that have the same kind of isotope, but other isotopes may be referenced indirectly by using a shift ratio to relate to a direct reference.

The “NMR Instruments” section contains two table that allows the user to add descriptions of the NMR probe and spectrometer that were used during the experiments. To achieve this the user adds a new specification for the appropriate kind of instrument then double-clicks to fill in the details for each of the rows that appears in the table.

Caveats & Tips

An experiment may be linked temporarily with a new shift list; selecting “<New>” in the Shift List column of the first tab then reseting the shift list back to the original one, in order to make a shift list that contains only chemical shift value for that experiment at that time. Without any experiment links these chemical shift values will not alter as peaks and assignments change.

References

A nomenclature and data model to describe NMR experiments. Fogh RH, Vranken WF, Boucher W, Stevens TJ, Laue ED. J Biomol NMR. 2006 Nov;36(3):147-55 (link)

Main Panel¶

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