Resonance Assignment/Abacus
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INTRODUCTION TO ABACUS
ABACUS (Applied BACUS) is a novel approach for protein structure determination that has been applied successfully for more than 20 NESG targets. ABACUS is characterized by use of BACUS, a procedure for automated probabilistic interpretation of NOESY spectra in terms of unassigned proton chemical shifts based on the known information on "connectivity" between proton resonances. BACUS is used in both the resonance assignment and structure calculation steps. The ABACUS is distinguished from conventional approaches to NMR structure determination mostly by its resonance assignment strategy (see Fig.1.1A).
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Figure 1.1. A.(on the left) Flowchart of resonance assignmnent by ABACUS. B. (on the top)Schematic description of two types of molecular fragments: traditional spin-system (AA-fragment) include all the atoms belonging to the same residue; PB-fragment includes all the atoms from one residue except the backbone amide group, plus the amide group from the next residue in the protein
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1)Lemak A., Steren, C., Arrowsmith, C.H. and Llinás, M. (2008) J. Biomol. NMR, 41, 29-41. 2) Grishaev, A., Steren, C.A., Wu, B., Pineda-Lucena, A., Arrowsmith, C. and Llinás, M. (2005) Proteins, 61,36-43. 3)Grishaev, A. and Llinás, M. (2004) J. Biomol. NMR, 28, 1-10.
Some features /advantages of the ABACUS protocol:
- It does not rely on sequential connectivities from less sensitive experiments such as HNCACB indispensable for most traditional sequential assignment procedures;
- Inter-residue sequential connectivities are established mainly from NOE data, which saves time at a later stage in “troubleshooting” NOE and resonance assignments.;
- Probabilistic nature of the ABACUS procedure provides measure of reliability of assignments, and therefore one can obtain a partial, yet highly reliable assignment (even when the NMR data are sub-optimal) with the knowledge of where to focus manual intervention;
- It can make use of partial spin-systems;
- It can efficiently identify manual errors in the input peak lists;
NMR spectra required for ABACUS
The spectra typically needed for ABACUS approach are most conveniently separated into 3 groups: NH-rooted, the CH-rooted and the aromatic (also CH-rooted). Table 1 shows the optimal set of NMR spectra. This, of course, is neither an exclusive or exhaustive list. For example, a simultaneous CN-NOESY could be recorded instead of three different ones listed in the table. In case there are very few aromatic residues in a protein, to collect only one aromatic spectrum, namely aromatic NOESY, could be enough for assignment of aromatic resonances.
Table 1. ABACUS optimal set of experiments
NH-rooted
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CH-rooted
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Aromatic
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15N-HSQC
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13C-CT-HSQC
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13C-HSQC-aro
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HNCO
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13C-HSQC
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H(C)CH-TOCSY-aro
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HNCA
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H(C)CH-TOCSY
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(H)CCH-TOCSY-aro
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CBCA(CO)NH
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(H)CCH-TOCSY
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13C-NOESY-HSQC-aro
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HBHA(CO)NH
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13C-NOESY-HSQC
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15N-NOESY-HSQC
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CCCONH-TOCSY (optional)
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H(CCCO)NH-TOCSY (optional)
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'Spin-system identification strategy
The resonance assignment procedure starts from grouping resonances in spin systems (PB-, or peptide bond, fragments) comprising correlated resonances from the side chain of residue i and the NH resonances of residue i+1 (see Figure1.1B). The uncompleted HN-rooted PB spin-systems, which include resonances of atoms only, are called bPB-fragments in this manual.
Spin-system identification in ABACUS approach consists of 3 main steps.
1. On the first step, bPB-fragments are collected from high sensitivity NMR correlation experiments (such as HNCO, CBCA(CO)NH, and HBHA(CO)NH) that transfer magnetization via the intervening peptide bond (see Figure 1.2A)
2. On the second step, completion of bPB-fragments with side-chain aliphatic resonances as well as identification of additional spin-systems (lacking HN resonances) is performed using HCCH-TOCSY and 13C-NOESY spectra (see Figure 1.2B)
3. Finally, spin-system validation and correction is performed. This step allows one to find mistakes made during spectra peak-picking and to correct the mistakes by going back to the spectra.
For each spin-system, 20 scores S(T) were calculated during the validation (see Figure 1.3). Here T corresponds to amino acid type, and T=A,R,D,…, and V, respectively. The score evaluate goodness-of-fit of the spin-system resonances to those observed in BMRB data base. If the best score , where , is too low, it means that either the spin-system has very unusual chemical shifts or the spin-system does not make sense and need to be corrected.
Fragments assignment by FMC
Sequence-specific assignment of PB-fragments is achieved using a Fragment Monte Carlo (FMC) stochastic search procedure. The scoring function used in the FMC procedure is based on both fragment amino acid typing (matching the spin system to amino acid types) and fragment contact map (reflecting which residue is next to which) derived from HNCA data and the analysis of NOEs interpreted by BACUS (see Figure 1.4).
Figure 1.4. PB-fragments mapping onto protein sequence.
Set of PB-frsagments: F1 F2 F3 F4 ....
Positions: protein sequence recycle bin
1 2 3 …… … Ns… .Ntotal
Scoring function:
Where
A is any possible fragment’s mapping (assignment) onto protein sequence;
Nf is number of PB-fragments;
Ns is number of residues in protein sequence;
Ntotl is number of total positions for fragment mapping; Positions in the “recycle bin” are reserved for discarded (not assigned) PB-fragments.
FMC procedure performs probabilistic assignment of PB-fragments. The assignment probabilities are calculated by Simulated Annealing (SA) or Replica Exchange Method (REM) Monte Carlo (MC) simulations. Here, is a probability of fragment k to occupy position s;k = 1,….,Nf. ; and s = 1,….,Ns+1. Sequence-specific assignment of PB-fragments is achieved by analyzing probabilities (see Figure 1.5) as well as sub-optimal fragment’s mapping that are provided by MC simulations.
FMCGUI
FMCGUI is a graphical interface that assist user to carry out resonance assignment and structure calculation using ABACUS approach.
FMCGUI_2.2 COMMANDS
0. FMCGUI objects.
Most of FMCGUI commands operate mainly with the following three objects that are located in computer memory: protein sequence, peak list, and PB-fragments.
Protein sequence. This object can be created in memory using [Data>Protein sequence>load] or [Project>load] commands.
The position ID of the first residue in the sequence should be specified by user upon loading sequence file (in the case it is not specified in the input file). Some commands in FMCGUI implies that the first residue of the protein sequence has position ID of 1. Therefore, if there is HIS-tag in the loaded sequence, it should be numbered accordingly starting with a negative position ID of the first residue.
Peak Lists. Different peak lists objects can be created in memory using [Data>”Peak list name”>load] or [Project>load] commands. For some peak-lists, peaks in the list could be referenced by spin-system (fragment) user ID.
The following table shows what peak lists are required referencing (+), peak lists
that are optionally referenced (+/-), and peak lists for which referencing is not used
even if present in the input file (-):
N15 NOESY
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-
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C13 NOESY H2O
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-
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Arom NOESY
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-
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N15 HSQC
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+
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C13 HSQC
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+
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HNCA
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+/-
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HNCO
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-
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CBCACONH
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+/-
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HBHACONH
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+
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List of Fragments. This object can be created in memory using [Fragment>Load], [Fragment>Create], or [Project>load] commands. Each fragment in the list has the following main properties:
- Fragment ID assigned by user, U_id;
U_id can’t be changed within FMCGUI.
- Assignment ID, A_id, that indicate the sequence position ID to which the fragment is assigned; ( A_id = -99 if the fragment is not assigned to any position in the sequence)
A_id could be set up or modified by the commands: [Assignment>Fix Assignment>Manually], [Assignment>Fix Assignment>Using probability Map], and [Assignment>Fix Assignment>Reset all].
- Typing probabilities , where t correspond to one of 20 AA residue types.
could be calculated or modified by the commands:
[Fragment>Type>Calculate] and [Fragment>Type>Fix].
- Three Fragment contact maps , , and , respetively. Each contact map scores the possibility for any fragment f to be next to fragment U_id in the protein sequence; were f and U_id stand for fragment user ID.
is calculated from HNCA spectrum by the command [Assignment>Contact>HNCA]
Fragment contact maps and , calculated from NOESY spectra with and without using BACUS procedure, respectively;
can be calculated by the commands [Assignment>Contact>NOE>fawn] and [Assignment>Contact>NOE>abacus], while is calculated by [Assignment>Contact>NOE>abacus].
- Fragment assignment probabilities and are calculated using SA and REM Monte Carlo simulations, respectively. Here s stands for protein sequence position ID.
is calculated by command [Assignment>Calculate probabilities>SA] or it can be loaded in memory using command [Assignment>Load probabilities];
is calculated by command [Assignment>Calculate probabilities>REM] or it can be loaded in memory using command [Assignment>Load probabilities];
The current values of all these properties for a particular fragment could be observed in the “Fragment Graph “window which is opened by command [View>Fragment]
1. Main window
The main frame of FMC Graphical Interface consist of 4 sections (see Figure )
- the title bar displays the name of the current project and the directory where the project is located;
- the bar with six menu: Project, Data, Fragment, Assignment, Structure, and View, respectively;
- the main message window, where message from the last executed command is displayed ;
- the log window, where the history of executed commands is shown.
[Project>New] : To 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] : To 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.
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 the case when the ID is not specified in the input file. It is recommended, that if there is His-tag in the sequence file, than the first residue ID should be set to a negative number so 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>load/Save as]
[Data>C13 NOESY>load/Save as]
[Data>Arom NOESY>load/Save as]
[Data>N15 HSQC>load/Save as]
[Data>C13 HSQC>load/Save as]
[Data>HNCA>load/Save as]
[Data>HNCO>load/Save as]
[Data>CBCACONHN>load/Save as]
[Data>HBHACONH>load/Save as] : To 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.
[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);
[Fragment>Load>PB fragments] : To load unassigned spin-systems in the memory.
Input format :
PB-fragments in standard format.
[Fragment>Save>PB fragments] : 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).
[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”.
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 AA types are generated from user-identified spin-systems. Each generated bPB-fragment is evaluated by a score SpS that measure how good the spin-system chemical shifts match corresponding statistical chemical shifts derived from BMRB database. 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.
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, 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-approvedbPB-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 AA types are generated from user-identified spin-systems. Each generated PB-fragment is evaluated by a score SpS 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 bPB-fragments.
Spin-system which have all SpS scores less than 10-4 are reported in the main FMCGUI window.
Following these warnings user can accept or to modify generated PB-fragments in the left section of “Create Fragment’ window. 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:
- 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.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 top section of FPM window user can select AA residue type t1.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 )
[Assignment>Contacts>HNCA] :To calculate fragments contact map.
Prerequisites:
- PB-fragments are loaded in memory ;
- typing probabilities are calculated;
- HNCA peak list (recommended to be referenced) is loaded in memory.
- tolerances are specified.
In the result, the contact map is calculated and loaded in the memory.
[Assignment>Contacts>NOE>fawn] :To calculate fragments contact map.
Prerequisites:
- PB-fragments are loaded in memory;
- typing probabilities are calculated;
- N15_NOESY peak list is loaded in memory.
- tolerances are specified.
In the result, the contact map is calculated using N15 NOESY peak list and loaded in memory.
[Assignment>Contacts>NOE>abacus] :To calculate both and fragments contact maps.
Prerequisites:
- PB-fragments are loaded in memory;
- typing probabilities are calculated;
- N15_NOESY and C13_NOESY peak lists are loaded in memory.
- tolerances are specified.
In the result, the contact map is calculated using only N15_NOESY peak list while contact map is calculated using both N15_NOESY and C13_NOESY peak lists that are interpreted by BACUS procedure. Both calculate maps are loaded in memory.
[Assignment>Calculate Probabilities>SA] :To calculate assignment probabilities.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments are loaded in memory;
- typing probabilities are calculated;
- fragments contact map is calculated;
- at least one of and fragments contact maps is calculated;
Probabilistic mapping of PB-fragments onto protein sequence is performed using Simulated Annealing Monte Carlo simulations.
A new window “Calculate SA” is open were user can specify different parameters in the control file of the SA simulations.
The main parameters to consider are:
- “Name of the SA run”. Normally the name is sa_run#. A new directory under this name will be created within PROJECTNAME/assign directory. SA calculations will be curried out and the results will be stored in this directory.
- “Size of the pool for unassigned fragments”. The number of positions that are appended to the protein sequence and discarded (unassigned) fragments, if there are any, will be located there. It is safe to over-estimate this number. (If this number is under-estimated, this will force the mapping of spurious spin-systems onto protein sequence);
- “Number of SA trajectories”. The time needed for calculations is proportional to this number. On the other hand, having more SA trajectories the assignment probabilities could be calculated more accurately. In the case of good data, when all SA trajectories converge to assignments with the same energy, 10-15 trajectories should be enough. In the case of poor data, it is better to calculate 40-50 SA trajectories.
- “NOE bbcmap type”. User should specify which one NOE contact map, (abacus) or (fawn) should be used in the calculations;
- “Fixing position flag”. If the flag is set to 1, sequence position of all fragments which has assignment ID > -99 will be fixed during the simulation.
- “Final temperature”. Setting the optimal final temperature will provide all SA trajectories converge to optimal or sub-optimal assignments (the assignments that are in the vicinity of the global energy minimum). The optimal final temperature could be find by running one or a few SA runs with 3-4 trajectories and by analysing convergence of the trajectories from the report shown in the main FMCGUI window after each run.
.
In the result of the SA calculations assignment probability map is
calculated and loaded in memory. The map is also saved in the file 'sa.probmap' located into PROJECTNAME/assignment/sa_run# directory.
[Assignment>Calculate Probabilities>REM] :To calculate assignment probabilities.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments are loaded in memory;
- typing probabilities are calculated;
- fragments contact map is calculated;
- at least one of and fragments contact maps is calculated;
Probabilistic mapping of PB-fragments onto protein sequence is performed using Replica Exchange Method Monte Carlo simulations.
A new window “Calculate REM” is open were user can specify different parameters in the control file of the REM simulations.
The main parameters to consider are:
- “Name of the REM run”. Normally the name is rem_run#. A new directory under this name will be created within PROJECTNAME/assign directory. REM calculations will be curried out and the results will be stored in this directory.
- “Size of the pool for unassigned fragments”. The number of positions that are appended to the protein sequence and discarded (unassigned) fragments, if there are any, will be located there. It is safe to over-estimate this number. (If this number is under-estimated, this will force the mapping of spurious spin-systems onto protein sequence);
- “Number of REM steps”. The time needed for calculations is proportional to this number. On the other hand, with more REM steps more extensive sampling of assignment space wil be achieved, which in turn results in more accurate assignment probabilities. This number should be increased for large proteins.
- “NOE bbcmap type”. User should specify which one NOE contact map, (abacus) or (fawn) should be used in the calculations;
- “Fixing position flag”. If the flag is set to 1, sequence position of all fragments which has assignment ID > -99 will be fixed during the simulation.
- “Low temperature”. The optimal low temperature will provide extensive sampling of should sub-optimal assignments during REM simulation.
User can check a correct setting of the low temperature as well as the number of REM steps by analysing a report shown in the main FMCGUI window after the calculations are done.
In the result, the 50 lowest energy assignments are used to calculate assignment probabilities . The probabilities are loaded in memory and saved in the file ‘rem.prbmap’ located into PROJECTNAME/assign/rem_run# directory.
[Assignment>Fix Assignment>Using Probability map] :To perform sequence specific assignment of PB-fragments using results of SA or REM calculations.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments loaded in memory;
- at least one SA or REM calculations of assignment probabilities was done
Calculation of assignment probabilities with FMCGUI could be repeated a few times using different methods and parameters. Results of each calculation are stored in a separate directory with the user-specified name. Therefore, there could be a few different directories (for example, sa_run1, sa_run2, rem_run0, rem_run1, rem_run2) located within PROJECTNAME/assign/ directory that contain different assignment probability maps.
User will be asked to select the calculation directory (sa_run# or rem_run#) and to specify the value of acceptance probability P_a. Normally, P_a =0.9 is appropriate. A fragment is considered to be assigned to a sequence position if the corresponding assignment probability (taken from the selected directory) is >= P_a.
In the result, part of the PB-fragments will be assigned, namely, their assignment IDs A_id will be specified. The assignment report will be shown in the main FMCGUI window as well as saved in the corresponding simulation directory (‘sa.fix ‘or ‘rema.fix’ files). For each sequence position, the IDs of both unambiguously and ambiguously assigned fragments are shown in the report. The list of discarded fragments is also presented.
[Assignment>Fix Assignment>Manually] :To perform sequence specific assignment of PB-fragments manually.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments are loaded in memory;
This command allows user to fix sequence position of individual fragments.
“Fragment Property Modification” window will be open. User can select a fragment in the bottom section of the window and the ingormation regarding the fragment assignemts will be displayed in this section. Namely, the graph shows assignment probabilities ( or ) that are currently loaded in memory and the text part shows the chemical shifts making up the fragment and it’s assignment ID (A_id).
To modify the current fragment assignment user have to select sequence position on the graph (by clicking on it by mouse) and then to press ‘Update’ button.
In the result, Assignment ID of the selected fragment will be set to the selected sequence position.
There are two special positions “U” and “B” shown on the graph at the end of the protein sequence.
Selecting “U” and pressing “Update” button results in changing assignment status of the fragments to Unassigned, that is A_id is set to -99.
Selecting “B” and pressing “Update” button results in fixing fragment position in the pool of discarded fragments.
[Assignment>Fix Assignment>Reset all] :To change assignment status of all fragments to “Unassigned”.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments are loaded in memory;
In the result, for all fragments A_id is set to -99.
[Assignment>Load Probabilities] :To load assignment probabilities in memory.
Prerequisites:
- protein sequence is loaded in memory;
- PB-fragments are loaded in memory;
- SA / REM calculations of assignment probabilities was done
User have to select a directory where SA or REM calculations were done (sa_run# or rem_run#).
The assignment probabilities from the selected directory will be loaded in memory.
[Structure>Constraints>Talos>calculate] :To generate dihedral angle constraints.
Prerequisites:
- protein sequence is loaded in memory;
- assigned shemical shifts are loaded in memory;
Backbone dihedral angles are predicted using TALOS and then transformed to dihedral angle contraints.
In the result, the constraints are saved in two formats: files 'belok.aco' and 'prot_dihe.tbl' for CYANA and CNS calculations, respectively. Both files are saved in the root project directory PROJECTNAME.
[Structure>Constraints>Talos>load] :To load dihedral angle constraints in CYANA format (aco-file).
[Structure>Constraints>H-bonds>Specify] :To prepare H-bond distance constraints manually.
Prerequisites:
- protein sequence is loaded in memory;
A new window “O/HN Pairs” is opened.
For each H-bond constraint, user have to specify O-HN pair by typing in the ID of residues corresponding to O and HN atoms. Pressing “OK” will save H-Bond constraints in two formats: files 'hbond.upl' and 'prot_hbond.tbl' for CYANA and CNS calculations, respectively. Both files are saved in the root project directory PROJECTNAME.
[Structure>Constraints>H-Bonds>load] :To load HBond distance constraints in CYANA format (upl-file).
[Structure>Calculate>Cyana] :To set up a new structure calculation run with CAYANA.
Prerequisites:
- protein sequence is loaded in memory;
- assigned chemical shifts are loaded in memory;
- N15_NOESY, C13_NOESY, and Aron_NOESY peak lists are loaded in memory
- Specified tolerances.
- dihedral angle constraints are created ( file “belok.aco” is present in the root project directory PROJECTNAME)
Optional:
- Hbond distance constraints (file “hbond.upl” is present in the root project directory PROJECTNAME)
- file that contains ZN ion ligands (file “zn_ligands” is present in the root project directory PROJECTNAME)
A new directory under the name that is specified by user (normally “crun#”) will be created inside project root directory PROJECTNAME. This directory contains all files required to start automatic structure calculations with CYANA.
[Structure>Calculate>Cyana] :To set up ABACUS structure calculations.(Under construction)
[Structure>RPF>RP] :To perform Recall/Precision analysis of structural ensemble.
Prerequisites:
- protein sequence is loaded in memory;
- assigned shemical shifts are loaded in memory;
- N15_NOESY, C13_NOESY, and Arom_NOESY peak lists are loaded in memory
- Specified tolerances.
- coordinates of structural ensemble in CYANA format (final.pdb) or in CNS format (prot_ref_al.pdb). pdb
The parameters to set up:
- “RP directory name”. Normally the name is rp#. A new directory under this name will be created within PROJECTNAME/assign directory. The results of the RPFanalysis will be stored in this directory.
- “sequence gap”. Residue pairs separated by less than the value of sequence gap will be excluded from generating expected peak lists.
- “cutting distance for recall”. Distance threshold for evaluating matching of an experimental peak to a structural ensemble (Recall score)
- “cutting distance for precition”. Distance threshold for generating expected peak from structural ensemble (Precision score)
The results of the RPF analysis will be saved in a new directory (the name of which is specified by user) that is located in the project root directory PROJECTNAME. The results include peak lists in the SPARKY format of both false negative and false positive peaks for different NOESY spectra in a separate files.
[Structure>RPF>DP] :To set up calculations of DP score with AutoStructure.
Prerequisites:
- protein sequence is loaded in memory;
- assigned shemical shifts are loaded in memory;
- N15_NOESY, C13_NOESY, and Aron_NOESY peak lists are loaded in memory
- Specified tolerances.
- coordinates of structural ensemble in CYANA format (final.pdb) or in CNS format (prot_ref_al.pdb). pdb
[Structure>Water refinement>calculate] :To set up water refinement of structural ensemble obtained with CYANA.
Prerequisites:
- structure calculation with CYANA should be done
Optional:
- RDC data in PALES format.
- file that contains ZN ion ligands (file “zn_ligands” )
User will be asked to specify the name of directory for water refinement calculations WATDIR and to select a number of files with coordinates and constraints. In addition to this user have to indicate cisProline residues (if there are any) and to specify protonation state of HIS residues which is double protonated by default.
In the result, a new directory WATDIR will be created inside PROJECTNAME directory that contains all files and scripts required to carry out water refinement calculations with CNS. It is recommended to start calculations on linux cluster.
[Structure>Water refinement>summary] :To create a summary.
Prerequisites:
- water refinement with CNS should be done.
User will be asked to specify residues used for structure superposition and to select the water refinement directory WATDIR.
The refined structural models will be superimpose and combined in one file. The created summary reports values of different energy components and constraint violation statistic for each structural model. A new directory WATDIR_results will be created. The directory contains final superimposed coordinates, distance and dihedral angle constrains in a format suitable for PDB deposition, summary and constraint violations report.
[Structure>Add ZN ligands] :To create zn_ligands file.
User have to tipe in IDs of residues that ligate zinc ion into the popped up entry window “ZN ligands”. If there are a few zinc ions, information for each ion should be provided in a separate row.
The file “zn_ligands” will be created inside the project root directory PROJECTNAME by pressing “OK” button.
[Structure>RCI] :To calculate Random Coil Index.
Random Coil Index is calculated using rci_v_1c.py script. New directory PROJECTNAME/rci that contains the results of calculations is created.