Resonance Assignment/Abacus/Sequence specific assignment of PB fragments

 Step 1. Peak picking of NOESY spectra

         To facilitate peak-picking of NOESY spectra, you can first generate expected tocsy peaks of PB-fragments using FMCGUI:  

•      open project PRJ2 { Project>Load }
•      generate tocsy peaks of N15 NOESY spectrum { Fragment>Expected Peaks>N15NOESY }
•      generate tocsy peaks of C13NOESY spectrum { Fragment>Expected Peaks>C13NOESY }

       Then, using SPARKY, read the expected peaks into corresponding spectra and complete peak picking manually.

 

Step 2. Probabilistic assignment of PB fragments to protein sequence

According to FMC procedure (see Introduction) you have to do the following:

1.     Probabilistic typing of PB-fragments
2.     Calculate  two fragments contact maps C_NOE and C_{HNCA .} C_NOE is a contact map based on 15N_ and 13C_NOESY data (it could be calculated by 2 different methods, “abacus” and “fawn”, respectively) and C_HNCA is a map based on HNCA data.
3.    Calculate assignment probabilities by Simulated Annealing (SA) or Replica Exchange Method (REM) Monte Carlo simulations

 

     This can be done by the following commands:    

•    open project PRJ2 { Project>load }
•    load 15N NOESY peak list { DATA>N15 NOESY>load }
•    load 13C NOESY peak list { DATA>C13NOESY H2O>load }
•    set tolerances  { Data>Tolerances }
•    calculate typing probabilities for all fragments { Fragment>Type>calculate>abacus }

        You have to consider the warning messages shown in the project main window and to analyze/modify typing probabilities manually by using “Fragment Property Modification” (FPM) window. To open FPM window use command { Fragment>Type>fix }. This window has 3 sections. Top section allows you to select a fragment (by user ID) and modify its typing probabilities. The middle section shows typing probabilities that correspond to the selected amino acid type for all fragments. Here you can fix for any fragment it’s  typing probability corresponding to the selected amino acid type to the values of 1 or 0.

 

•   calculate fragment contact map from HNCA data { Assignment>Contacts>HNCA }         

      Contact map C_HNCA is calculated and loaded in the memory. It is strongly recommended to check the messages in the project main window regarding HNCA peak list. In case there are inconsistencies present in the input HNCA peak list, go back to spectra and fix the list. 

•   calculate fragment contact map from NOESY data { Assignment>Contacts>NOE>abacus }. In the result, two contact maps, C_{NOE_B} and C_{NOE_F} are calculated and loaded in the memory. Calculation of C_{NOE_B}  makes use of BACUS procedure for NOESY data interpretation, while calculation of  C_{NOE_F}   does not. 
• <span />Calculate assignment probabilities.    It can be done using two different Monte Carlo simulation methods. Namely, Simulating Annealing (SA) { Assignment>Calculate Probabilities>SA } and Replica Exchange Method (REM) { Assignment>Calculate Probability>REM }. Before starting calculations you have to specify control parameters. The main parameters to consider are: ‘Name of the SA/REM run’, ‘Size of the pool for unassigned fragments’, ‘number of SA runs’, ‘Final Temperature’ (SA), ‘Low Temperature’ (REM), and ‘NOE contact map’. The results of the calculations will be stored in the directory under specified name which is created inside PRJ2/assign/ directory. The main result consists of optimal and sub-optimal fragments assignments and assignment probability map. The last one will be automatically loaded in the memory as well.

         Calculation of assignment probabilities could be repeated a few times using different methods and parameters. Result of each calculation is stored in a separate directory. Therefore, there could be a few different directories (for example, sa_run1, sa_run2, rem_run0, rem_run1, rem_run3… ) within PRJ2/assign/ directory that contain different assignment probability maps.

 

Step 3. Sequence-specific assignment of PB fragments by analyzing probabilities

 

   A fragment assignment to a sequence position using FMCGUI could be done in two ways, manually and using assignment probability map, respectively.

 

•    manual assignment { Assignment>Fix Assignment>manually }.

This command pops up ‘Fragment Property Modification’ window. You can change the assignment ID of any selected fragment using the bottom section of the window.

 

•   assignment using probability map  { Assignment>Fix Assignment>using probability map }. You have to select a calculation directory (sa_run# or rem_run#) that contains assignment probability map, , and to specify the probability threshold . A fragments k will be assigned to position s if the condition    is satisfied (see Figure 1.4).

 

Step 4. Assignment analysis

 

In the case of poor data, only a part of the fragments get assigned unambiguously. The uncertainty in fragments assignment could be resolved manually with the help of FMCGUI command [View>Assignment]. This command pops up “Assignment Graph” window that provides you with graphical tool to visualize the current assignment and to analyze sub-optimal fragment assignments (see Figures 2.21-2.25).

 

Step 5. Final resonance assignment

 

When sequence specific PB-fragment assignment is done you have to put in order assigned fragments and to assignCO resonances using command [Fragment>Modify assigned].