Run MARS from Analysis

Installation

In order to run MARS from Analysis you have to have to have MARS installed on your computer system. You can find information about MARS on the MARS website and download Mac and Linux versions from the MARS Download section. Make sure you follow the setup instructions to add

    setenv MARSHOME    /your_install_path/mars_install_directory/bin

    alias runmars            $MARSHOME/runmars

to your .cshrc file.

You will also need to install PSIPRED. You can download it from the PSIPRED Download page. Once you have extracted the tar.gz file, follow the instructions in the README file to compile the program. Then make sure you add 

    setenv PSIPRED_DIR    /your_install_path/psipred

to your .cshrc file.

Note that MARS probably won't work if your CCPN project is saved on a server or external hard drive - it has to be located on your local computer.

Once you have done these things, Analysis will recognise the MARS installation. You can check this by opening the Automated Seq. Assignment popup through M: Assignment: Automated Seq. Assignment. The {Automation} tab will show you whether Analysis can find your MARS and PSIPRED installations or not. If not, there will be a warning message:

If the installation can be found the MARS options and [Run MARS] button will be shown:

Instructions

Setup

Go to the {Settings} tab to set basic input parameters (as shown in the example figure below) for the automated sequential assignment:

• • Select the Peak Lists to include in the automatic assignment procedure in the ‘Peak Lists’ panel.

  • To include or exclude a peak list from the procedure, simply toggle the |Use?| parameter.

  • Set the peak matching tolerances in ‘Peak-Peak Match Tolerances’

  • Make sure the correct Chain and Shift List are selected under 'General'

Finding resonances from peaks

The next step is to go to the {Spin Systems} tab and let Analysis automatically find resonances from the peak lists and the experiment types, and to assign them to the most likely atom types.

To do this, go to the {Spin Systems} tab, which at first presents us with a table listing all the spin systems in the selected Shift List. To find resonances and assign them to the most likely atom types, click [Find Resonances From Peaks]. After confirmation, Analysis sorts the peak lists and starts making its assignments. After a few seconds, a list of Intra and Inter residue resonances is presented:

What is important here is that Analysis comments under |Comments| where it has found potential problems in the procedure in how to find and assign resonances. Start to inspect a problem by selecting the related spin system, and click [Show Peaks] or [Go To Root Location]. Problems could, for instance, be multiple CA or CB resonances.

Note that it isn't necessary to have assigned resonances with CA or CB atom types to the carbon dimension of your spectra. But doing so, will result in fewer errors. On its own, Analysis is not very good at identifying Ser and Thr CA/CB resonances correctly, or CB resonances with chemical shifts between 40 and 50ppm, so you are likely to have to make some corrections by hand for these spin systems in order for the programme to run well.

Automation

The next step is to go to the {Automation} tab and set parameters for the automation using MARS:

See the MARS website for details of what effect changing some of these parameters might have. Note that you will be able to use more features and options (e.g. including RDC or coupling data) if you run MARS natively. See 'Running MARS externally' below for more information on how to do this.

Click [Run MARS] to let the program run and make its automatic sequential assignment predictions.

After some time the assignment results and their reliability is plotted versus the sequence in the {Score Graph} tab:

Inspect and confirm predictions, and commit assignments

To inspect and confirm predictions, go to the {Predictions} tab. In this tab, the residues are listed, with their |Prediction Score|, |Best Matching Spin System| and |Confirmed Spin System| etc..

To inspect predictions, you can display strips of the sequentially assigned spin systems by selecting residues of interest, setting the category of matching in the lower left pulldown menu named “Any Available” and click [Strip Selected Using:]. The window for display is chosen in “Window:” in the {Settings} tab.  

To commit assignments, select the assignments that you think are correct and click [Confirm Selected]. To add the confirmed assignments to the project, click [Commit Assignments].

Notes

MARS will only run if you have not yet assigned your protein. You can run MARS iteratively, assigning some parts, running it again with the 'Keep existing assignments?' option checked and then gradually improving your assignment. However, if you have largely assigned your protein and then try running MARS without the 'Keep existing assignments?' option checked, you are not likely to get very good results. Unfortunately, the program won't just run as though you hadn't made any assignments. If you are unsure of any assignments, de-assign your spin systems in the Spin Systems popup (M: Resonance: Spin Systems) and then run MARS again.

Running MARS externally

If you want to run MARS externally in order to take advantage of the increased number of options and data that can be used, it is possible to export your chemical shifts and create the other MARS input files required.

File export

You can use the Format Converter (M: Other: Format Converter) to export your project in MARS format. From the Format Converter menu select M: Export: Mars. Then chose the 'Project Export Menu'. Here you can select the location and name of your project file (call this mars.inp), the directory where all the files are written to, as well as the Chain and Shift List you want to use. Click on [Export project file] to export all your data.

Your will then have the mars.inp file with all your parameters which you can change as you like, a cs.tab file containing your chemical shifts and fastaSequence.tab file containing your protein's sequence. All you then need to run MARS is your secondary structure prediction file in PSIPRED format which you can obtain from the PSIPRED webserver.

For more details on how to run MARS see the MARS website.

Notes

The Format Converter will only export the chemical shifts of spin systems not assigned to a chain, i.e. your protein needs to be unassigned for this to work.

To ensure you have all the intra- and inter-residue chemical shifts correctly assigned, it may be worth running the [Find resonances from peaks] routine in the {Spin Systems} tab of the Automated Seq. Assignment popup before exporting your chemical shifts (see above).