Resonance Assignment/Abacus/FMCGUI commands

 

2. Project Menu

 

Project>New

:Start a new project.

 User have to provide a name of the project PROJECTNAME,and to select a directory that will host the project. The project root directory with the same name PROJECTNAME is created.

             

Project>Load

: Continue to work on previously saved project.

 

User have to select file PROJECTNAME.prj in the directory PROJECTNAME, where PROJECTNAME is the name of the root directory of the project.

 

Project>Save

 : To save the current state of the project.

 

What is currently in the computer memory is saved in the file PROJECTNAME.prj located in the root directory of the project.

 

Poject>Quit 

To save the current state of the project and to quit.

 

3. Data Menu

The DATA section serves to load&save the data (such as protein sequence and peak lists). Since there are different formats of data-files that could be loaded in memory or saved on disk, one can use this section as format converter as well.

 

Data>Protein Sequence>Load 

: To load a protein sequence into memory.

 

 

The input formats:

                             -   1-letter code (fasta format);

                             -   3-letter code (standard format).

 

User have to select the file with sequence and to specify the first residue ID in case it is not specified in the input file.

If there is His-tag in the sequence file, it is recommended to set the first residue ID  to a negative number such that the first residue of a protein has ID of 1.

 

Data>Protein Sequence>Save as

: To save protein sequence in the file on disk.

 

The output formats:

                               - 1-letter code (fasta format);

                               -  3-letter code ("standard" format, for cyana)

                               -   3-letter code (for AutoStructure);

                               -  3-letter code (for RCI);

 

There are separate buttons for different peak lists.

 

Data>N15 NOESY>

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Data>Arom NOESY>

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Data>N15 HSQC>

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Data>HBHACONH>

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 :   Load or save a peak list.

 

Input and output formats: 

                                        - Sparky;

                                        - Xeasy;

                                         - Standard

                                      

Data>Tolerances

: To set tolerances for chemical shift matching in different spectral dimensions.

 

4. Fragment menu

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Fragment>Load>assigned

: To load assigned chemical shifts (spin-systems) in the memory. 

 

Prerequisites:

      - Loaded sequence

 

 

Input formats:

                      - assigned AA-fragments in standard format;

                      - CYANA chemical shift file (prot-file);

 

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Fragment>Load>PB fragments

: To load unassigned spin-systems in the memory.

 

Input format :

                        PB-fragments in standard format.

 

 

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: To save PB-fragments in a file on disk.

 

Output format:

                       PB-fragments in standard format.

 

The name of the saved file and it’s location are specified by user.

There are 3 options to save PB-fragments in the file:         

   - in order of fragments index, that is in the order by which fragments are stored in memory;

   - in order of fragments user ID, U_id;

   - in order of fragments assignment ID, A_id. In this case 2 files are saved. One file, with user    specified name 'user_name', contains only fragments assigned to protein sequence positions, that is to positions with residue ID of >= 1. The second file, with the name 'user_name_na', contains all not assigned fragments (that is fragments with A_id = -99).

 

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Fragment>Save>cyana

: To save assigned chemical shifts (that is fragments with A_id >0 ) in CYANA format.

 

Fragment>Save>bmrb

: To save assigned chemical shifts in the format suitable for BMRB deposition (star2.1)

 

 Fragment>Save>talos

 : To save assigned chemical shifts in the format suitable for TALOS/CS-Rosetta;

 

  Fragment>Save>abacus

:To save unassigned PB-fragments in the format suitable for BACUS;

 

 

Fragment>Create>fawn

: To create/evaluate bPB-fragments.

 

Prerequisites: 

- loaded in memory referenced peak lists of CBCA(CO)HN, HBHA(CO)HN, N15HSQC, and HNCA spectra;

- Specified tolerances.

                                      

There are two steps in executing this command.

On the first step, a fake C13HSQC peak list is created and shown in the popped up window “fake C13HSQC” (see Figure 2.2)

 

 

 

 

 

 

 

 

 

 

                             

 

 

 

 

 

 

 

User can use the information shown in the main FMCGUI window and check /edit the list in the entry section of “fake C13HSQC” window.  Pressing OK will result in loading the peak list from the entry window into memory as C13HSQC peak list.

 

On the second step, a number of bPB-fragments corresponding to 20 different amino acid types are generated from user-identified spin-systems. Each generated bPB-fragment is evaluated by a score S(T)  that measure how good the spin-system chemical shifts match corresponding statistical chemical shifts derived from BMRB database (see Figure 1.2). The bPB-fragment with highest score is selected to form a list of bPB-fragments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In the result, a new window ‘Create Fragment’ pops up and warning messages of the ‘sps_create’ script are shown in the main FMCGUI window (see Figure 2.3).

The window consists of three sections. The left sections contains suggested bPB-fragments, while the other sections contains two reports of fragments scoring with both C and H resonances and with only C resonances, respectively. Following the warning messages shown in the main FMCGUI window (see Figure 2.4), user can accept/modify generated bPB-fragments. Alternatively, when ‘poor’ bPB-fragments are present, user can go back to spectra, fix the pick lists accordingly, and repeat the fragment generation again.

User-approved bPB-fragments will be loaded in the memory by pressing OK button.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Results:

-          C13HSQC peak list loaded in memory

-          bPB-fragments are loaded in memory

 

 

[Fragment>Create>abacus] : To create/evaluate PB-fragments.

 

Prerequisites: 

- loaded in memory referenced C13HSQC, N15HSQC, and HNCA peak lists and  not referenced CBCA(CO)HN peak list; ( as an option, HNCA peak list could be not referenced as well)

- Specified tolerances.

 

On the second step, a number of PB-fragments corresponding to 20 different amino acid types are generated from user-identified spin-systems. Each generated PB-fragment is evaluated by a score S  (see Figure 1.2) that measure how good the spin-system chemical shifts match corresponding statistical chemical shifts derived from BMRB database. The PB-fragment with highest score is selected to form a list of PB-fragments.

Spin-system which have S_max less than 10^-4 are reported in the main FMCGUI window (see Figure 2.4). Following these warnings user can accept or to modify generated PB-fragments in the left section of “Create Fragment’ window (see Figure 2.5). Alternatively, user can go back to spectra, fix the pick lists accordingly, and repeat the fragment generation again.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 User-approved bPB-fragments will be loaded in the memory by pressing OK button

 

Results:

-          PB-fragments are loaded in memory

 

[Fragment>Type>Calculate>fawn/abacus] : Probabilistic typing of bPB-fragments (fawn) or PB-fragments (abacus) .

 

Prerequisites: 

- loaded in memory protein sequence

- loaded in memory PB-fragments

- specified tolerances.

 

Results:

- Fragment typing probabilities are calculated and loaded in memory.

 

The main FMCGUI window displays (see Figure 2.6):

-          the summary table that shows how many fragments of each AA-residue type are expected and how many fragments were actually recognized by the typing script;

-          warning messages that suggest user to check and possibly modify typing manually of some fragments manually

 

      

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[Fragment>Type>fix] : To modify typing probabilities .

 

Prerequisites: 

- loaded in memory protein sequence

- loaded in memory PB-fragmen

 

New window "Fragment Property Modification" (FPM) window is opened (see Figure 2.7). This window has 3 sections. 

 

 

In the top section of FPM window user can select fragment user ID, U_id. Then typing probabilities  for all AA types t will be shown on the graph. The chemical shifts of the fragments and its assignment status (A_id) are shown as well. User can modify typing probabilities of the selected fragment by selecting AA types by clicking right mouse button and pressing ‘Update’ button. In the result only propapbilities corresponding to the selected AA types will be set to the same non-zero values.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In the middle section of FPM window user can select amino acid residue type t''₁(see Figure 2.8). Then the graph will show typing probabilities    that correspond to the selected residue type t₁ for all available fragments IDs f . Selecting a particular fragment U_id by clicking right mouse button (the color of U_id is changed to red) and pressing “Update” button will set the probability  to 1 while  for all other f will be set to 0. Selecting a particular fragment U_id by clicking left mouse button (the color of U_id is changed to blue) and pressing “Update” button will set the probability  to 0.

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[Fragment>Expected Peaks>”spectra name”] : To generate different peak lists expected from covalent structure of fragments.

 

Prerequisites: 

- protein sequence is loaded in memory

- PB-fragments are loaded in memory

 

[Fragment>Modify assigned] : To correct assigned fragments.

 

Prerequisites: 

- loaded in memory protein sequence

- loaded in memory PB-fragments

- loaded HNCO, CBCACONH, and HNCA peak lists.

- specified tolerances.

 

 

In the result  CO chemical shifts are added and chemical shift names are corrected for PB-fragments which are assigned (that is which has A_id > -99 )