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HomoScope

HomoScope is CARA's tool for homonuclear 2D assignment. It can also be used to work with heteronuclear 2D spectra.

Homonuclear Assignment

The following assumes you have homonuclear 2D NOESY/COSY/TOCSY spectra and that you follow the standard procedure for homonuclear assignment as outlined in the book by Kurt Wüthrich, NMR of Proteins and Nucleic Acids, 1986, Wiley ISBN -471-82893-9.

1. Preparing the definitions: Go to Spectrum Types ... , click on the little arrow on the left of the 2D-NOESY definition. You will see the definitions for both dimensions. Now right-click on the first dimension, and select Add Label... from the context menu and enter "HN" into this case. Do the same for the second dimension, but enter "HA". These labels will be used as typical spin-system starting labels. Repeat these steps for your TOCSY / COSY definitions.
2. Starting and extending a SpinSystem?: Now open HomoScope and use Pick New System (Shortcut PY) on any HN/HA cross peak. You should definitely use also View->Show List to have the developing SpinList? available during assignment. If you use the TOCSY to extend your spin system, don't use PY again but use EH - Extend Horizontally and EV- Extend Vertically to add Spins to your System. Trivial Peaks can be added by using Propose Peak ... (PP) or depending on circumstances Propose Horizontally / Propose Vertically. You can review the recommended strategy on pages 130ff of Kurt Wüthrich's book, you will find that HomoScope is closely modeled upon it.
3. Sequence-specific assignment: First of all you can use the typical patterns as defined by Wüthrich's book (page 136ff). If your system has HN, HA, HB2, HB3 it is classified as AMX, HN, HA1, HA2 it is AX (more commonly called Gly) and so on. You can use Assignment-> Set System Type to select these. If the list is empty, your template does not contain these definitions, so you will have to add them quickly (see below). If you are certain about the which Amino Acid the spin system is you can use Assignment -> Set Assig. Candidates... to select the correct one. You can also record negative information in this way. For example if you have an HN you know it's not a Pro, so you can use "Assignment -> Set Assig. Candidates..." select All and then deselect Pro to record this. You do not need to set both Assignment Candidates and System Types. CARA takes account of either of these inputs. However setting incompatble selections is bad, since no assignment possibility will then be found.
4. Linking Spin-Systems via sequential NOEs: If you see a sequential NOE crosspeak between System 3 and system 5 choose Assignment-> Link Systems... and enter 3 and 5. Be careful about the order. If you have a little fragment, with some System Types or Amino Acids identified, you can use Assignment-> Show Alignment to Map it onto the Sequence. Recommended procedure is to continue adding to the fragment until only a single possibility remains. It is not recommended to pick the best out of several. You can right-click onto a proposed fragment in the Alignment window and choose Assign from there. Little black dots will indicate those spin systems that are already assigned. Note that CARA also use CA/CB chemical shifts if they are available in the spinlist to rank the different possible fragments (e.g. You can get the CA and CB shifts from assigned natural abundance spectra).
5. Linking aromatic rings to their backbone spin-system: The aptly named Eat System... function does this automatically. You Right-Click the backbone system in the SpinList? and select Eat System. Then enter the System number of the aromatic ring system.
6. Adding the Spin System Types: again see page 136ff. Go to Spin System Types in the CARA-Explorer and right-click the empty list, choose New Type ... and enter the Name, e.g. AMX or AX. Do this for all 10 types. Very often nowadays we work in H2O, so it is easier to define Gln, Glu, Met, Arg and Lys as a single type called long and Pro as a separate type called Pro instead of the recommended way of the K.Wüthrichs book which assumes D2O as solvent. You can also add the 4 aromatic sub-types, if you want. Now you have to go to the list of Amino Acids in the ResidueTypes pane of CARA-Explorer. Right-click on each Amino Acid and use Set System Type to select the correct one; e.g. long for Gln, Glu, Met, Arg, Lys and so on.

Heteronuclear Assignment

• Check assignments: HomoScope combines assigned spins from the SpinList? into pairs expected from the SpectrumType and displays them in the current spectrum . E.g. a [1H,13C]-HSQC can be used to verify assignments based on spectra which do not correlate the two spins. (like HA coming from HNHA and CA coming from HNCA).
• Complete assignments: If one spin of a pair is known, visual inspection of a spectrum can be used to find the partner spin. HomoScope provides tools to facilitate this, like visual rulers and chemical shift matching.
• Find unpicked spins or systems: You may pick a spin pair in HomoScope and then use CARA's other tools like SystemScope to extend this spin system and possibly merge it into an existing one (with eat system command in the SpinList?). This is one method to find Prolines that could not be assigned during backbone assignment.

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