Getting started with CARA for protein backbone assignment

  1. Make sure you have an account set up with NMRBox, and connect to one of the VMs following their instructions. https://nmrbox.nmrhub.org/
  2. You can run cara from the command line with: 
    $ cara
  3. When you run cara from the NMRbox command line, it will give you an option of several templates, or you can import your own following http://wiki.cara.nmr.ch/ImportingTemplate 
    What template should cara open with?
   [0] - /usr/software/cara/Templates/NucleicAcids1.2.cara
   [1] - /usr/software/cara/Templates/ProteinLiquidState1.9v1.cara
   [2] - /usr/software/cara/Templates/ProteinSolidState1.0v3.cara
   [3] - None
  4. If you have your protein sequence as a fasta file eg. GDAVKREA… save it with the .aa extension eg. sequence.aa, and cara has a script to convert it to CARA format (eg sequence.seq) in the CARA terminal.
  5. Go to Scripts in CARA, right click on OneLetterFileToSeqFile, select “Execute…”, choose your sequence file, and enter a new name for your newly formatted sequence file, like myprotein_cara.seq. This file will appear in the directory from which you launched CARA, and it looks like this:
GLY 1
ASP 2
ALA 3
VAL 4
LYS 5
ARG 6
GLU 7
ALA 8
  1. If that option in CARA fails, you can use the perl script one2three.pl on the command line to convert your sequence, available on NMRbox in /public/kedmonds/scripts
  2. Make sure your new file with the 3-letter sequence ends in “.seq.”
  3. Right-click on “Projects” on the left side and select “Import project from sequence…” and select your file.
  4. Now a “+” appears next to Projects, and when you click on it, you can see your new project. You probably want to right click on the name of the project to rename it.
  5. You might want to renumber your sequence, if you’re working on a C-terminal domain, or you want to be consistent with existing work. You can click on “Sequence” to see the sequence you just imported. If you didn’t have numbers in the file, it starts with 1. You can right-click on a residue and select “Renumber from here…”, and choose the appropriate number for that residue. Unfortunately, this only changes the chain number, and not the ID. It turns out that you really want the ID to match the chain number, so I wrote a script to fix that. It’s not a lua script, so you can’t run it from within CARA, so you’ll have to save your repository (File -> Save As…), and quit CARA. Run the script on your old cara file to make a new one:

/public/kedmonds/scripts/cara-scripts/ModifyCARAresidueIDs.py -i oldfile.cara -o newfile.cara

  1. After converting the sequence file you should create a new project from sequence… following this tutorial: http://wiki.cara.nmr.ch/CreateNewProject
  2. Follow this tutorial to import your HSQC, HNCO, HNCA, HNCOCA, HNCACO, CBCACONH, HNCACB spectra http://wiki.cara.nmr.ch/ImportSpectra . You can have multiple spectra of each type, and you should have at least one HSQC that was recorded in the same session on exactly the same sample as your 3D(s).
  3. Open the HSQC that goes with your HNCO in the polyscope by right-clicking on the spectrum and then selecting Open with Polyscope. Oddly, the best explanation of what we want to do is in steps 1-5 of the Synchroscope tutorial: http://wiki.cara.nmr.ch/SynchroScope . But first try out points 4-10 of http://wiki.cara.nmr.ch/SpectrumNavigation for how to move the cursor and zoom in the HSQC.
  4. Right-click in the right-most strip of the PolyScope and select the HNCO.
  5. Place the cursor on a strong peak in the HSQC.
  6. The right-most strip should show a 2D slice of the 3D HNCO peak, with the vertical dimension being carbon frequencies and the horizontal dimension being nitrogen frequencies. The middle strip shows a slice through the same cursor position, but with the horizontal dimension being proton frequency instead of nitrogen.
    1. If you don’t see a peak in the strips, you may have to do some troubleshooting – check your processing and especially chemical shift referencing, center frequencies and sweep widths. You may need to make 2D projections of your 3D spectra if it is especially tricky.
  7. Right-click in the right-hand strip and choose “set peak width”, and set it to 3, to get a rounder peak (displaying a wider portion of the N sweep width.
  8. Do the same thing in the next strip to the left, and set it to 0.3 to get a rounder perspective on that peak, displaying a narrower portion of the H sweep width.
  9. Fortunately you only have to do this once per spectrum if you save your work.
  10. Ultimately you will need to do the same procedure (points 6-11) for your other 3D spectra, but let’s dive in with the HNCO for now.
  11. Assuming you see a peak in the strips whenever you click on an HSQC peak, your HNCO is probably processed and loaded correctly.
  12. To calibrate the HNCO relative to the HSQC, follow step 4 of this tutorial, still using the Polyscope: http://wiki.cara.nmr.ch/SynchroScope . If this is tricky, you may have to resort to step 5.
  13. You should have one peak in the HNCO for every peak in the HSQC, if that peak represents the amide NH of a residue. There may also be tryptophan, glutamine, and asparagine sidechain peaks in your HSQC that might not always have peaks in the 3D. You can now pick some systems and spins.
  14. Select the system you picked for calibration in the plane. Pick a “spin” in the 3D by right clicking on the center of the main peak in the strip and selecting “pick label”, then choosing “C -1” representing the carbonyl carbon of the preceding residue.
  15. Now right click in the HSQC frame and select “show 3D plane”. With the peak still selected, use shift and arrow keys to move the cursor in the HSQC plane to the center of the HNCO peak at the carbon frequency you selected in the strips. When the cursor is in the right place select “calibrate” from the “Plane” menu.
  16. Follow the same procedure to reference the other 3D spectra.
  17. Find “Pick 2D peaks” from the scripts tab in the project frame along the left side of the main cara window, and run that script.

Note:

Peaks are observed in each NMR experiment for backbone assignment:

  1. HNCO: HN(i) and CO(i-1)
  2. HNCaCO: HN(i) and CO(i) and CO(i-1)
  3. HNCOCa: HN(i) and Ca(i-1)
  4. HNCa: HN(i) and Ca(i) and Ca(i-1)
  5. CbCaCoNH: HN(i) and Ca(i-1) and Cb(i-1)
  6. HNCaCb: HN(i) and Ca(i) and Ca(i-1), Cb(i) and Cb(i-1) or it can be collected with only Cb’s and glycine Ca’s.

Backbone assignment scheme:

https://protein-nmr.org.uk/solution-nmr/assignment-theory/triple-resonance-backbone-assignment/

 

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