Smartnotebook Example 3: hsqc-orb

Many spectroscopists will have homologous sequence data and they will constantly refer to these shift values while disseminating nmr data. Here is your chance to encorporate this shift information into your shift constraints file in a reasonable fashion. In this example we are going to use our homologous Calmodulin and TnC shifts to construct a more accurate constraints file for our sequence, skeletel TnC using a piece of software called orb. 

	> cd hsqc-orb
	> snbview 
	When nmrview comes up, type "snb" at the nmrview console.

  1. In this example, snb has already been initialized with a BMRB default shift constraints file. You will see one chain, Fig 1 please click the "Best Fit" button and you will see that Gly 21 Hn has a Test Stat of 2.88, just barely acceptable for the purposes of assignment. Now let's what happens when we try to formulate a shift constraints file based on prior information. Fig 2

  2. Click "Configure / Update" in the Options menu. Click the item "Initialize snb" and press "Execute". Fig 3

  3. Smartnotebook comes up in initialization mode. The first 3 screens you are familiar with, just press "Continue".

  4. For the 4th screen, we click the radiobutton "Generate shift constraints from homologous sequence shift files." Press "Calculate". Fig 4

  5. At this point you will see the "Welcome to Orb" window. Orb has its own standalone program, paper, software history and web page . At the time of this software release, the orb documentation is not up to date for its usage in smartnotebook. However the way the software works hasn't changed so you will still find useful information here. Fig 5

  6. Orb now asks for your homologous chemical shift files to be located in a single directory in PPM format. We have these files located in a directory called data.ppm, so just press "Continue". Fig 6 Click here if you are interested in seeing an example of PPM format.

    Note: If you had shift files in star format, you can click the second button. Enter your data directory of star files. Click "Predict" and view your output. Then click "Continue". I am looking for more examples to test star to PPM file conversion.

  7. The next step is to create a sequence alignment file. Select the "Create alignment file using xalign" radiobutton and press "Continue". Fig 7 At this point you a placed in the hands of another software program called xalign .

  8. In order to be shielded from the complexity of creating an input file, select the "Please generate a default input file" button. Upon clicking this button, xalign tries to guess your sequence file and you will see that it is set to "./cdc4p.seq". You will have to tell xalign where your homologous shift files are located and so for the "PPM directory" field please enter "data.ppm". Fig 8 At this point you can press "Calculate" and your input file is generated for you. You can press the View button to see this file if you want. Fig 9 We are done so press "Continue".

  9. For the Output Specification window, the options here should not matter much, just press Calculate. Fig 10 View the multiple alignment output and make sure the computer generated alignment is reasonable. Press the "Dismiss" button. Fig 11 If the output is not correct, you will have to read the orb help files on generating a better alignment. Since we are happy with this alignment, we press "Exit" to leave xalign.

    Note: If your homologous sequences have less homology than the example we are presenting, it is difficult to say if creating a constraints file with orb is worth it.

  10. Ok, so we are finally have our inputs ready to run orb. Press "Continue". Fig 12 Make sure the Alignment File field has "snb.out/xalign.out" and your Shifts directory has "data.ppm". When you press "Verify Data", orb does indeed find your 2 homologous shift files of "TnC" and "CaM". Press "Continue". Fig 13

  11. In this window we are ready to run orb. The only thing we need to set is the sequence to predict. Go to "Sequence to Predict" and change the item from TnC to 'cdc4p'. At this point you could click on "Options" to get an inkling of the complexity which goes into trying to make a shift predict. Otherwise, press "Predict". Fig 14 "Dismiss" the output window when you are done and press "Exit" to leave orb. Fig 15 Finally we are back in smartnotebook with our new shifts file in "./snb.out/orb.shifts.ppm". Press "Continue". Fig 16

  12. Press "Finish Init" in the last window. Does this shift file make a big difference? Well, sometimes it does. If you hit the "Best Fit" button now, you will see that we have a much better prediction for Gly 21 Hn and now the Test stat has dropped to 0.50. But certainly if we had more homologous shifts, it would help alot. Fig 17

    Making your own shifts file is not easy, even with the help of orb and xalign. I think it could be worth it when your assignment task is difficult and you rely on those homologous chemical shifts to help make correct decisions.

Smartnotebook Examples

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