Resonance Assignment/Abacus/Sequence specific assignment of PB fragments

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Contents

 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:  
       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 you have to do the following:
  1.     Probabilistic typing of PB fragments
  2.     Calculate  two fragments contact maps CNOE and CHNCA . CNOE is a contact map based on 15N_ and 13C_NOESY data (it could be calculated by 2 different methods, “abacus” and “fawn”, respectively) and CHNCA 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:    
        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.
 

      Contact map CHNCA 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, CNOE_B and CNOE_F are calculated and loaded in the memory. Calculation of CNOE_B  makes use of BACUS procedure for NOESY data interpretation, while calculation of  CNOE_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.
 
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.
 
 

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.
 

Step 5. Final resonance assignment

 
When sequence specific PB fragment assignment is done you have to put in order assigned fragments and to assign CO resonances using command { Fragment>Modify assigned }.
          

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