As described in the papers:

  1. Mars - robust automatic backbone assignment of proteins (2004) Young-Sang Jung and Markus Zweckstetter, J. Biomol. NMR, 30, 11-23.
  2. Backbone assignment of proteins with known structure using residual dipolar couplings (2004) Young-Sang Jung and Markus Zweckstetter, J. Biomol. NMR, 30, 25-35.
  3. Automatic assignment of the intrinsically disordered protein tau with 441-residues (2010) Narayanan RL, Dürr UH, Bibow S, Biernat J, Mandelkow E, Zweckstetter M, J. Am. Chem. Soc., 132, 11906-11907.


Contact: mzwecks@gwdg.de


What is MARS ?
Download
Setup
Getting Started
Setting up input files
Setting up assignment parameters
How to run MARS
Output
Analysis of assignment / Testing
Directory structure
Important points to remember
For advanced users

What is MARS?

    MARS is a powerful program for robust automatic backbone assignment of proteins. MARS is applicable to proteins with extreme chemical shift degeneracy such as high molecular weight proteins and partially disordered or fully intrinsically disordered proteins. MARS is successfully used in many labs world-wide.

    MARS is interfaced with the program CCPNmr enabling direct easy access to automatic assignment without export or re-import of data.

    MARS assignment results can also be read into the program SPARKY. This allows visual validation of the assignment results. Thus, several cycles of automatic assignment using MARS and manual validation on the screen can be performed, in order to complete assignment even in difficult cases.





    Key features

    1. MARS works with a wide variety of NMR experiments, from 3D triple-resonance experiments up to the most complex 6D and 7D APSY experiments.
    2. MARS is interfaced with CCPNmr Analysis enabling direct easy access to automatic assignment without export or re-import of data.
    3. MARS results can be directly read into the program SPARKY. Thus, several cycles of automatic assignment and manual validation can be performed.
    4. MARS allows to fix sequential connectivity, residue-specific as well as residue-type assignments.
    5. MARS enables the use of 3D structures, through the calculation of residual dipolar couplings and scalar couplings, for improved resonance assignment.
    6. MARS is applicable to both deuterated and protonated proteins.
    7. MARS offers unsurpassed accuracy for proteins with incomplete data.
    8. MARS has excellent tolerance against erroneous chemical shifts.
    9. MARS combines secondary structure prediction with statistical chemical shift distributions, which are corrected for neighboring residue effects


Download


    The MARS software is freely available for academic users.

    Industry and commercial users are invited to obtain a license at MARS Commercial license.


    MARS (version 1.2)

      Suse Linux 2.4.20
      Mac OSX


      The download provides a compressed tar archive with a MARS executable and example files. The archive can be unpacked with a command like the following: 
        
             tar -xvzf mars-1.2_linux.tar.gz
      NOTE: The Suse Linux version should also work on RedHat Linux.


    Graphical user interface for MARS (version 1.0)

      There is also a simple graphical user interface for MARS available:
      LINUX/MAC


      PALES (home)

      For assignment using residual dipolar couplings the software PALES has to be installed in addition. This is only required if a 3D structure is known.

    Setup

      Open your .cshrc files in your home directory. Add the three lines below to your .cshrc file. The setenv LC_NUMERIC command is only required in case you have a German Unix version.

      
	setenv MARSHOME   directoryName

	alias runmars     directoryName/runmars

        setenv LC_NUMERIC "C"
      

	setenv PALESHOME  directoryName
      (directoryName is the name of the directory that contains the binary and script files.) 
      

        An example:



	setenv MARSHOME    /home/spine/local/MARS/mars-1.0_linux/bin

	alias runmars      $MARSHOME/runmars

        setenv LC_NUMERIC  "C"
      The MARS GUI may be started by executing one of the following files from a X11 window: 
      

        marsgui-1.0-linux (for LINUX)

        marsgui-1.0-MAC or marsgui-1.0-MAC-AQUA (for MAC)


    Getting started

      Input


      MARS is a program for backbone assignment of 13C/15N labeled proteins. Accordingly, following input is required:

      • Optional

        • table that allows restriction of the amino acid type and/or fixing of an assignment
        • table that allows fixing of sequential connectivities between pseudoresidues.

      How to run Mars

      1. Prepare your chemical shift table.
      2. Get your primary sequence in FASTA format.
      3. Get a secondary structure prediction using the Psipred web server.
      4. Adjust the parameter setup file (mars.inp).
      5. Type 'runmars mars.inp'

      Output

      • Assignment result filtered for high, medium and low reliablity ('assignment_AA.out').

      • Assignment result including alternative assignments that show up with a 10 % probability ('assignment_AAs.out').
      • The most likly assignment for each pseudoresidue ('assignment_PR.out').
      • Summary of all possible connectivities ('connectivity.out').
      • Summary of reduced possible connectivities ('connectivity_reduced.out').
      • Chemical shift tables with updated assignments that can be read into SPARKY ('sparky_all.out, sparky_CA.out, sparky_CA-1.out, sparky_CB.out, ...').

      • Detailed information about predicted chemical shifts, number of reliable assignments, number of constraints for each pseudoresidue, matrices matching experimental and back-calculated chemical shifts and/or RDCs and pseudoenergy matrices at each iteration step ('mars.log').

    Setting up input files

      Obligatory


      1. A Mars run is controlled by the parameter setup file (mars.inp). This has to be adjusted to the available experimental data. Please see below for a detailed description of the parameters.

          Lines with a '#' sign as first character as well as empty line are ignored.
          Do not change the variable names such as nIter. 
           mars.inp(MARSHOME/example/noStructure/1ubq/input/mars.inp)

              
            

                fragSize:     5               # Maximum length of pseudoresidue fragments



                cutoffCO:     0.25            # Connectivity cutoff (ppm) of CO [0.25]

                cutoffCA:     0.2             # Connectivity cutoff (ppm) of CA [0.5]

                cutoffCB:     0.5             # Connectivity cutoff (ppm) of CB [0.5]

                cutoffHA:     0.25            # Connectivity cutoff (ppm) of HA [0.25]

                cutoffHN:     0.02            # Connectivity cutoff (ppm) of HN [0.15]

                cutoffN:      0.05            # Connectivity cutoff (ppm) of N [0.10]



                cutoffnHN:    0.02            # Connectivity cutoff (ppm) of HN+1 [0.15]

                cutoffnN:     0.05            # Connectivity cutoff (ppm) of N+1 [0.10]



                fixConn:      fix_con.tab     # Table for fixing sequential connectivity

                fixAss:       fix_ass.tab     # Table for fixing residue type and(or) assignment



                pdb:          0               # 3D structure available [0/1]

                resolution:   NO              # Resolution of 3D structure [Angstrom]

                pdbName:      NO              # Name of PDB file (protons required!)

                tensor:       NO              # Method for obtaining alignment tensor [0/1/2/3/4]

                nIter:        NO              # Number of iterations [2/3/4]

                dObsExh:      NO              # Name of RDC table for exhaustive SVD (PALES format)

                dcTab:        NO              # Name of RDC table (PALES format)



                deuterated:   0               # Protonated proteins [0]; perdeuterated proteins [1]



                rand_coil:    0               # Folded proteins [0]; disordered proteins [1]



                sequence:     1ubq_fasta.tab  # Primary sequence (FASTA format)

                secondary:    1ubq_psipred.tab # Secondary structure (PSIPRED format)

                csTab:        1ubq_cs.tab     # Chemical shift table
          Optional parameters: 
           

              
            

                cyssTab:     cys.tab          # Table listing oxidized cysteines

                csPred:      1                # Theoretical chemical shifts from MARS [1/0]
      2. chemical shift table

          The chemical shift table follows the SPARKY format. It consists of a header, pseudoresidues and chemical shifts. The header has to be defined before the listing of chemical shift values starts and includes the variable names for the chemical shifts. Currently 10 different chemical shifts are supported and should be indicated by 'Ca', 'Ca-1', 'Cb', 'Cb-1', 'CO', 'CO-1', 'HA', 'HA-1', 'H' and 'N'. These variable names have to be in the same order as the columns for the different chemical shifts. The first column has to be the pseudoresidue column and other columns are chemical shift columns. Pseudoresidue means the name of the group of peaks which share the same (or similar due to the experimental imperfection)  N and HN chemical shifts. Lines with a '#' sign as first character as well as empty lines are ignored. Missing chemical shift values have to be indicated by ' - '.

          
              1ubq_cs.tab(MARSHOME/example/noStructure/1ubq/1ubq_cs.tab)

              

              
            

                 N CO-1 H CA-1 CA

  PR_2    123.220 170.540 8.900 54.450 55.080

  PR_3?   115.340 175.920 8.320 55.080 -

  PR_4?   118.110 172.450 8.610 59.570 55.210

  PR_5GLY 121.000 175.320 9.300 55.210 60.620

  PR_6GLY 127.520 - 8.820 60.620 54.520

  PR_7    115.400 177.140 8.730 54.520 60.470

  PR_8    121.330 176.910 9.100 60.470 57.580

  PR_9    105.590 178.800 7.630 57.580 61.400

  PR_10?? 108.890 175.520 7.810 61.400 45.460

                 :

                 :

                 :
          Pseudoresidue names can consist of characters+number+characters and characters are optional. Pseudoresidues are distingushed by the whole set of characters. Therefore arbitrary names and numbers are allowed for pseudoresidues. (this is new in version 1.1.1)

          What to do if a chemical shift cannot unambigously attributed to a specific pseudoresidue?
          MARS does not perform peak picking, referencing of spectra or grouping of peaks into pseudoresidues (PRs). In our lab we use SPARKY to perform these tasks. This allows visual control and refinement of pseudoresidues. When manually inspecting PRs, amide degeneracy can often be resolved, as peak shapes and the higher resolution in a 2D HSQC spectrum can be taken into account. If HN/N overlap remains, multiple spin systems should be provided to MARS comprising the full set of possible combinations of peaks. In order to avoid an unreasonable high number of PRs in these cases, ambiguous peaks can also be partially discarded, as MARS does not favor pseudoresidues with more complete chemical shift information during the assignment process. The suspicious peaks can be reinserted when running MARS a second or third time, after an initial MARS run was performed, the assignment results were visually validated using SPARKY and verified assignments were fixed.

          Glycine: Fix the amino acid type in fix_ass.tab
          MARS does not favor pseudoresidues with more complete chemical shift information. Therefore, it might happen that a spin system belonging to a glycine is wrongly assigned to another amino acid type, because both the connectivity might be fullfilled and the chemical shift matches sufficiently well. To avoid these problems, it is useful to fix the amino acid type of the spin systems that have uniquely been identified as glycines in the fix_ass.tab table.



      3. The primary sequence of the protein has to be in FASTA format.

          IMPORTANT: 'X' and 'Z' can not be used for the characters of a sequence.

          
                  1ubq_fasta.tab (MARSHOME/example/noStructure/1ubq/1ubq_fasta.tab)

                  
            

                    > ubq

                    MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN

                    IQKESTLHLVLRLRGG
      4. Secondary structure prediction table

          This has to be in Psipred format. Use the Psipred web server to get the table.
          
                  1ubq_psipred.tab (MARSHOME/example/noStructure/1ubq/1ubq_psipred.tab)

                  
            

                    PSIPRED PREDICTION RESULTS Key Conf: Confidence (0=low, 9=high)

                    Pred: Predicted secondary structure (H=helix, E=strand, C=coil)

                    AA: Target sequence Conf: 968896699888999867863189999999997689875658887777738887136726

                    Pred: CEEEEECCCCCEEEEEECCCCCHHHHHHHHHHHHCCCHHHEEEEECCEECCCCCCHHHHC

                    AA: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYN

                    10 20 30 40 50 60 Conf: 8988889999950699 Pred: CCCCCEEEEEEECCCC

                    AA: IQKESTLHLVLRLRGG 70

        If a 3D structure and experimental RDCs are available:
      5. PDB file

          All standard PDB files can be used (including MOLMOL files).

          IMPORTANT: When using shape-prediction all atoms in the PDB file will be used including pseudo atoms (ANI).


      6. Dipolar coupling input

        Experimental dipolar couplings are supplied according to the PALES table format:

        • The protein sequence should be given as shown by one or more "DATA SEQUENCE" lines. Space characters in the sequence will be ignored.
        • The table must include columns for residue ID, three-character residue name and the atom name for both atoms that are involved in the dipolar coupling as well as the dipolar coupling itself, its error and a weighting factor. Segment ID and Chain ID are optional.
          IMPROTANT: The atom notation must match that of the PDB file.
        • The table must include a "VARS" line that labels the corresponding columns of the table.
        • The table must include a "FORMAT" line that defines the data type of the corresponding columns of the table.
        • Lines with a '#' sign as first character as well as empty lines are ignored.


          Example dipolar coupling table (excerpts): 
          DATA SEQUENCE MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL

DATA SEQUENCE EDGRTLSDYN IQKESTLHLV LRLRGG



VARS   RESID_I RESNAME_I ATOMNAME_I RESID_J RESNAME_J ATOMNAME_J D      DD    W

FORMAT %5d     %6s       %6s        %5d     %6s       %6s    %9.3f   %9.3f %.2f



    2    GLN      N      2    GLN     HN     -15.524     1.000 1.00

    3    ILE      N      3    ILE     HN      10.521     1.000 1.00

    4    PHE      N      4    PHE     HN       9.648     1.000 1.00

    5    VAL      N      5    VAL     HN       6.082     1.000 1.00



    1    MET      C      2    GLN     HN       3.993     0.333 3.00

    2    GLN      C      3    ILE     HN      -5.646     0.333 3.00

    3    ILE      C      4    PHE     HN       1.041     0.333 3.00

    4    PHE      C      5    VAL     HN       0.835     0.333 3.00



    1    MET      C      2    GLN      N       2.651     0.125 8.00

    2    GLN      C      3    ILE      N      -3.768     0.125 8.00

    3    ILE      C      4    PHE      N       1.463     0.125 8.00

    4    PHE      C      5    VAL      N      -1.726     0.125 8.00



    2    GLN      N      2    GLN     HN     -15.524     1.000 1.00

    3    ILE      N      3    ILE     HN      10.521     1.000 1.00

    4    PHE      N      4    PHE     HN       9.648     1.000 1.00

    5    VAL      N      5    VAL     HN       6.082     1.000 1.00



    1    MET     HA      1    MET     CA     -38.341     1.000 0.50

    2    GLN     HA      2    GLN     CA      11.662     1.000 0.50

    3    ILE     HA      3    ILE     CA      18.424     1.000 0.50

    4    PHE     HA      4    PHE     CA      26.733     1.000 0.50


      Optional

      1. The chemical shift of oxidized cysteines differs signifcantly from that of reduced ones. Therefore, the residue number of cysteines involved in a disulphide bond should be listed in a file (i.e. residues 3,23,36 and 58 are involved in a disulphide bond). Reduced cysteines should not be listed here. 
        
cys.tab (MARSHOME/example/noStructure/1ubq/cys.tab)

	
          

3

23

36

58
      2. The theoretical chemical shifts used by MARS for matching fragments to the primary sequence can be modified manually. This allows the usage of experimental chemical shifts from a homologues protein or using an improved chemical shift prediction software. User-supplied 'theoretical' chemical shifts can be included by: (i) Setting the csPred parameter in mars.inp to '1'. (ii) Run Mars to obtain a table with theoretical chemical shifts predicted by MARS (Cs_pred.tab). (iii) Modify Cs_pred.tab. (iv) Change the csPred parameter from '1' to '0' in mars.inp. (v) Run Mars again.

      3. When additional information such as specific amino acid type labeling or initial manual assignments are available assignment of pseudoresidues can be restricted to single or to a set of residues. The first column has to be a pseudoresidue name followed by residue numbers or amino acid types to which the assignment should be restricted. Assignments can be fixed one by one by specipying the corresponding residue numbers or restrict it to a whole residue fragment by specifying the starting and ending residue number (inclusive) connected by '-' (without a blank in between the start and end number!). At the same time, amino acid types can be fixed by specifying the corresponding one letter code. More than one amino acid type can be specified by concatenation of the corresponding one letter codes (i.e. attach additional one-letter codes without blank in between). 
        
fix_ass.tab (MARSHOME/example/noStructure/1ubq/fix_ass.tab)

	
          

PR_3 3

PR_10 10-15 23 34

PR_12 12 34-36

PR_13 13

PR_14 14 16 HKT

PR_15 LFR 66-69 13-16 9 71

PR_16 EVA
      4. Also sequential connectivities can be fixed. This is especially useful when assignment is done iteratively by Mars and manually. The first and second column are pseudoresidue names. The first column is the name of the pseudoresidue for which the intra-residual chemical shift can be connected to the inter-residual chemical shift of the pesudoresidue in the second column. 
         fix_con.tab

                (MARSHOME/example/noStructure/1ubq/fix_con.tab) 

                
          

                   PR_2 PR_3 PR_3 PR_4 PR_4 PR_5 PR_11 PR_12

                  PR_12 PR_13 PR_13 PR_14 PR_25 PR_26 PR_26 PR_27

    Setting up assignment parameters

      fragSize:

        Sequential connectivity is established by matching inter- and intra-residual chemical shifts. Fragments comprising up to fragSize pseudoresidues are searched for exhaustively. The maximum segment length fragSize is a compromise between the desired total execution time of a MARS assignment run and the ability to reliably place PR segments onto the protein sequence.

        According to our tests a fragSize of 5 is large enough to get reliable assignments (pseudoresidue fragments with length five can in most cases be placed uniquely into the protein sequence when intra- and inter-residual Ca and Cb chemical shifts are available).

        For smaller proteins or if more computing power is available larger fragment sizes (six or seven) can be employed. This is expected to be useful if, for example, no Cb chemical shift information is available.

      cutoff

        • cutoffCO is the tolerance value (ppm) for matching intra- and inter-residual chemical shifts of C'.
        • cutoffCA is the tolerance value (ppm) for matching intra- and inter-residual chemical shifts of Ca.
        • cutoffCB is the tolerance value (ppm) for matching intra- and inter-residual chemical shifts of Cb.
        • cutoffHA is the tolerance value (ppm) for matching intra- and inter-residual chemical shifts of Ha.

        Cutoff values should be determined according to the resolution of the spectra. If chemical shifts were obtained from standard HNCACB, CBCACONH and HNCO experiments reasonable values will be 
          Ex.)

	cutoffCO: 0.1

	cutoffCA: 0.5

	cutoffCB: 0.5

	cutoffHA: 0.1
        Note that too small error bounds will lead to a small number of reliable assignments.

      fixConn

        This is optional. If you want to fix sequential connectivities, prepare a table like fix_con.tab and specify the table name, otherwise set the fixConn parameter to NO.

        NOTE: At one iteration step MARS generates 60 assignment solutions and extracts reliable assginments from these solutions. After the first iteration step MARS automatically fixes reliable assignments and reliable sequential connectivities obatined from previous iteration steps without user intervention. The iteration is continued until the number of reliable assignments does not increase any more. Therefore, one can see fixed assignments and fixed sequential connectivities on the screen during a MARS run although the user didn't fix anything at the start of MARS. 
          Ex.)
          

		fixConn: NO



			or



		fixConn: fix_conn.tab

      deuterated

        If a protein is perdeuterated, set the deuterated parameter to 1. Otherwise put it to 0. 
          Ex.)
          

		deuterated: 0



			or



		deuterated: 1

      sequence

        Specifiy the name of the file that contains the primary sequence of your protein in FASTA format. 
          Ex.)
          

		sequence: 1ubq_fasta.tab

      secondary

        Specifiy the name of the file that contains the secondary structure information of your protein in PsiPred format. 
          Ex.)
          

		secondary: 1ubq_psipred.tab

      csTab

        Specifiy the name of the file that contains the experimental chemical shifts (SPARKY format). 
          Ex.)
          

		csTab: 1ubq_cs.tab



      If no 3D structure or RDCs are available,
      put the additional paramters as below:

       
        

pdb: 		0

resolution: 	NO

pdbName: 	NO

tensor: 	NO

nIter: 		NO



dObsExh: 	NO

dcTab: 		NO


      If a 3D structure and experimental RDC are available,
      following parameters have to be set up:

      pdb

        Put the pdb flag pdb to 1, in order to use RDCs and the known 3D structure (otherwise set it to 0 ). 
          Ex.)
          

		pdb: 1

      resolution

        Specify the resolution of your crystal structure. If you don't konw the resolution of the structure because it is a homology model, set the resolution to ~ 4.0. In this case it will be useful to perform multiple assignment runs with decreasing values for the resolution parameter (suggested range is 2.0 < resolution < 6.0). The optimum value corresponds to the assignment run where the maximum number of reliable assignments was obtained. 
          Ex.)
          

		resolution: 1.8

      pdbName

        Name of file containing the coordinates of the 3D structure. All standard PDB files (including Molmol) can be used. IMPORTANT: Protons have to be present. 
          Ex.)

		pdbName: 1ubq.pdb

      tensor

        Method for obtaining an initial estimate of the alignment tensor. Four different modes are available that can automatically be accessed by specifying 1, 2, 3 or 4. The standard mode is 3.

          If 1 is selected, MARS will use a gridSearch for estimating the orientation of the alignment tensor.
          If 2 is selected, MARS will use exhaustive back-calculation (exhSVD). (dObsExh parameter has to be setup!)
          If 3 is selected, MARS will use singular value decomposition (SVD).
          If 4 is selected, MARS will use shape-prediction (shapePred).

          Ex.)
          

		tensor: 3
          (It is recommended to use 1 or 3 for the tensor parameter. Modes 2 and 4 require additional knowledge or RDCs in nearly neutral alignment media.)

      nIter

        MARS refines the initial alignment tensor estimate (obtained by the tensor method specified above) several times using SVD based on the reliable assignments obtained in previous iteration steps. Here, the number of refinement steps of the alignment tensor, nIter, can be defined. According to our tests 2 refinement steps are enough. 
          Ex.)
          

		nIter: 2

      dObsExh

        For exhaustive back-calculation (tensor mode 2) an RDC table is required that contains RDCs of a specific amino acid type. If the tensor mode is 1, 3 or 4, put the dObsExh parameter to NO. 
          Ex.)
          

		dObsExh: NO



			or



		dObsExh: dObs_1ubq_GLY.tab

      dcTab

        Name of file that contains the experimental RDC values (in PALES format). 
          Ex.)
          

		dcTab: dObs_1ubq.tab



    Output

    1. The first column is the residue number of the protein; the second column is the pseudoresidue that the residue is assigned to. The third column indicates the degree of reliability of each assignment. Three levels of reliability are distinguished:

      H indicates high reliablity as defined in the MARS paper. M and L do not fulfill all the criteria required for H reliablity and the specific criteria employed are adjusted automatically according to the completeness of the input data. Please see below for the robustness of assignments labelled as M and L. F indicates assignments fixed by the user. 
      

assignment_AA.out

		
        

MET_1

GLN_2      PR_2 (M)

ILE_3      PR_3 (M)

PHE_4      PR_4 (H)

VAL_5      PR_5 (H)

LYS_6

THR_7

LEU_8

THR_9      PR_9 (M)

GLY_10     PR_10 (H)

LYS_11     PR_11 (H)

THR_12     PR_12 (H)

ILE_13     PR_13 (H)

THR_14     PR_14 (H)

LEU_15     PR_15 (H)

GLU_16     PR_16 (M)

VAL_17

GLU_18

PRO_19

SER_20     PR_20 (L)

ASP_21

THR_22

	:

	:

	:
    2. The first column is the residue number of the protein. Additional colums list pseudoresidues that can be assigned to this resdiue. Numbers in parenthesis are assignment probablities. Only pseudoresidues with an assignmnent probablity of higher than 10% are shown. assignment_AA.out is a subset of the assignments here. 
      

assignment_AAs.out

		
        

MET_1

GLN_2      PR_2 (96)

ILE_3      PR_3 (100)

PHE_4      PR_4 (100)

VAL_5      PR_5 (100)

LYS_6      PR_6 (63)  PR_8 (30)

THR_7      PR_7 (76)

LEU_8      PR_8 (61)

THR_9      PR_9 (100)

GLY_10     PR_10 (100)

LYS_11     PR_11 (100)

THR_12     PR_12 (100)

ILE_13     PR_13 (100)

THR_14     PR_14 (100)

LEU_15     PR_15 (100)

GLU_16     PR_16 (100)

VAL_17     PR_17 (73)

GLU_18

PRO_19

SER_20     PR_20 (86)

ASP_21     PR_21 (65)

THR_22     PR_57 (33)

	:

	:

	:
    3. assignment_PR.out lists the most likely assignment for each pseudoresidue present in the input chemical shift table. The first column is the pseudoresidue and the second is the residue (to which the pseudoresidue can be assigned to most likely). NOTE: 'The most likely assignment' does not mean reliable assignment and two pseudoresidues can also be assigned to one residue. The information present in assignment_PR.out is useful if a pseudoresidue is not assigned to any residue in assignment_AAs.out and one asks himself what it might be assigned to. 
      

assignment_PR.out

		
        

PR_2 	 GLN_2

PR_3 	 ILE_3

PR_4 	 PHE_4

PR_5 	 VAL_5

PR_6 	 LYS_6

PR_7 	 THR_7

PR_8 	 LEU_8

PR_9 	 THR_9

PR_10 	 GLY_10

PR_11 	 LYS_11

PR_12 	 THR_12

PR_13 	 ILE_13

PR_14 	 THR_14

PR_15 	 LEU_15

PR_16 	 GLU_16

PR_17 	 VAL_17

PR_18 	 GLU_18

PR_20 	 GLN_40

PR_21 	 GLN_41

PR_22 	 SER_57

	:

	:

	:
    4. In connectivity.out all possible sequential connectivities between pseudoresdiues are listed. All numbers are pseudoresidue numbers. The first column (closed by '-->') is the pseudoresidue number for which connectivities are listed. If no additional entries are present no connectivities could be found for that pseudoresidue. Otherwise, all pseudoresidue numbers are listed for which the inter-residual chemical shift can be matched to the intra-residual chemical shift of the pseudoresidue in the first column. 
      

connectivity.out

		
        

PR_2   -->	PR_3    PR_5    PR_35   PR_43   PR_69   PR_74

PR_3   -->	PR_4    PR_23   PR_30   PR_56

PR_4   -->	PR_3    PR_5    PR_29   PR_35   PR_43

PR_5   -->	PR_6    PR_8    PR_71

PR_6   -->	PR_2    PR_7    PR_49   PR_55

PR_7   -->	PR_6    PR_8

PR_8   -->	PR_9    PR_21

PR_9   -->	PR_10

PR_10  -->	PR_11   PR_48   PR_76

PR_11  -->	PR_12

PR_12  -->	PR_13

PR_13  -->	PR_14

PR_14  -->	PR_15

PR_15  -->	PR_16   PR_44

PR_16  -->	PR_17   PR_69   PR_74

PR_17  -->	PR_18   PR_60

PR_18  -->	PR_16

PR_20  -->	PR_9    PR_21

PR_21  -->	PR_22   PR_40   PR_41   PR_50   PR_73

PR_22  -->	PR_23   PR_56
    5. Detailed information about assignment parameters, percentage of expected intra- and inter-residual Ca, Cb, Co and Ha chemical shifts present in the input chemical shift table, predicted chemical shifts, number of reliable assignments, number of constraints for each pseudoresidue, matrices matching experimental and back-calculated chemical shifts and/or RDCs and pseudoenergy matrices at each iteration step. 
      

mars.log
        



------------------------------------------------------------------------------------------

fragSize: 	5   			# Maximum length of pseudoresidue fragments

cutoffCO: 	0.25			# Connectivity cutoff (ppm) of CO [0.25]

cutoffCA: 	0.2			# Connectivity cutoff (ppm) of CA [0.5]

cutoffCB: 	0.5			# Connectivity cutoff (ppm) of CB [0.5]

cutoffHA: 	0.25			# Connectivity cutoff (ppm) of HA [0.25]





fixConn: 	fix_con.tab		# Table for fixing sequential connectivity

fixAss: 	fix_ass.tab 		# Table for fixing residue type and(or) assignment





pdb: 		0   			# 3D structure available [0/1]

resolution: 	NO			# Resolution of 3D structure [Angstrom]

pdbName: 	NO			# Name of PDB file (protons required!)

tensor: 	NO	   		# Method for obtaining alignment tensor [0/1/2/3/4]

nIter: 		NO  			# Number of iterations [2/3/4]



dObsExh: 	NO			# Name of RDC table for exhaustive SVD (PALES format)

dcTab: 		NO			# Name of RDC table (PALES format)



deuterated: 	0 	  		# Protonated proteins [0]; perdeuterated proteins [1]

sequence: 	1ubq_fasta.tab 		# Primary sequence (FASTA format)

secondary: 	1ubq_psipred.tab	# Secondary structure (PSIPRED format)

csTab: 		1ubq_cs.tab        	# Chemical shift table

------------------------------------------------------------------------------------------



# of AA: 76

# of PRO: 3

# of GLY: 6

# of Assignable AA: 72

# of PR: 72



CA: 100.0   CB:   0.0   CO:   0.0   HA:   0.0

Ca: 100.0   Cb:   0.0   Co: 100.0   Ha:   0.0





   AC    RC     RW

--------------------

   53    11     0

--------------------

   55    13     0

   54    13     0

--------------------

   54    20     0

   53    20     0

--------------------

			:

			:

			:

			:

			:


    Analysis of assignment

      1. Check the connectivity.out to verify that your chemical shift table has been made properly. If there are problems in your chemical shfit table due to miscalibration of spectra and(or) many pseudoresidues grouped incorrectly, you will see many missing sequential connectivities in the connectivity.out table.

      2. MARS keeps reliablility of assignment even for highly degenerate and/or incomplete data sets and labels assignments according to three different levels of reliability, H (high reliable assignment), M (medium reliable assignment), L (low reliable assignment). The following table shows the robustness of Mars assignment for different proteins, different connectivity cutoffs and ranked according to H, M and L reliability. From this table it becomes clear that even medium reliable assignments are basically error-free and only for less stringent connectivity cutoffs assignments labelled as L contain a few errors.


          Table 1. (BMRB chemical shifts used for the test. / COcutoff: 0.25, CAcutoff: 0.5, CBcutoff: 0.5)
          Tested proteins Used chemical shfits # of AA (assignable AA) H (Correct/Incorrect) M (Correct/Incorrect) L (Correct/Incorrect)
          Maltose binding protein C', Ca, Cb
          723 (654)
          639/ 0
          9/ 0
          0/ 0
          Maltose binding protein Ca, Cb
          370 (335)
          303/ 0
          18/ 1
          0/ 0
          EIN of the phospoenolpyruvate Ca, Cb
          259 (248)
          232/ 0
          6/ 0
          0/ 0
          E-cardherin domains II Ca, Cb
          227 (167)
          76/ 1
          5/ 1
          12/ 9
          Human prion protein Ca, Cb
          210 (190)
          130/ 0
          9/ 0
          5/ 0
          Superoxide dismutase C', Ca, Cb
          192 (117)
          101/ 0
          2/ 1
          2/ 0
          Calmodulin/M13 complex C', Ca
          148 (144)
          37/ 0
          7/ 0
          17/ 2
          E. coli EmrE C', Ca, Cb
          110 (74)
          35/ 0
          6/ 0
          10/ 3
          Human ubiquitin Ca, Cb
          76 (72)
          72/ 0
          0/ 0
          0/ 0


          Table 2. (BMRB chemical shifts used for the test. / COcutoff: 0.15, CAcutoff: 0.2, CBcutoff: 0.4)
          Tested proteins Used chemical shfits # of AA (assignable AA) H (Correct/Incorrect) M (Correct/Incorrect) L (Correct/Incorrect)
          Maltose binding protein C', Ca, Cb
          723 (654)
          639/ 0
          9/ 0
          0/ 0
          Maltose binding protein Ca, Cb
          370 (335)
          324/ 0
          7/ 0
          2/ 0
          EIN of the phospoenolpyruvate Ca, Cb
          259 (248)
          246/ 0
          0/ 0
          0/ 0
          E-cardherin domains II Ca, Cb
          227 (167)
          102/ 0
          7/ 0
          19/ 0
          Human prion protein Ca, Cb
          210 (190)
          127/ 0
          4/ 0
          5/ 0
          Superoxide dismutase C', Ca, Cb
          192 (117)
          104/ 0
          0/ 0
          1/ 0
          Calmodulin/M13 complex C', Ca
          148 (144)
          142/ 0
          0/ 0
          0/ 0
          E. coli EmrE C', Ca, Cb
          110 (74)
          58/ 0
          4/ 0
          0/ 0
          Human ubiquitin Ca, Cb
          76 (72)
          72/ 0
          0/ 0
          0/ 0


          Table 3. (Raw peak lists used for the test. / CAcutoff: 0.3, CBcutoff: 0.5, HAcutoff: 0.05)
          Tested proteins Used chemical shfits # of AA (assignable AA) H (Correct/Incorrect) M (Correct/Incorrect) L (Correct/Incorrect)
          Z domain protein Ca, Cb, Ha
          71 (67)
          65/ 0
          0/ 0
          0/ 0
          Z domain protein Ca, Cb
          71 (67)
          34/ 0
          6/ 0
          2/ 0




      3. Connectivity_reduced.out filters all detected connectivities reported in connectivity.out according to high reliable assignments. This can be useful for manual refinement of a Mars assignment.





    Directory structure

      MARSHOME:

      

	$MARSHOME/bin

	$MARSHOME/example
      



	$MARSHOME/example/noStructure

	$MARSHOME/example/Structure
      



	$MARSHOME/example/noStructure/1ubq

	$MARSHOME/example/noStructure/1zym

	$MARSHOME/example/noStructure/1dmb
      



	$MARSHOME/example/Structure/1ubq

	$MARSHOME/example/Structure/1zym

	$MARSHOME/example/Structure/1dmb


    Important points to remember

      1. Spectra calibration and proper peak grouping are the most important points.


      2. When grouping inter- and intra-chemical shifts try to use the same spectrum for extraction of inter- and intra-chemical shfits of a given atom type.

        For example, when you want to get intra- and interresidual chemical shifts of Ca, extract both chemical shifts from the HNCA spectrum. Only take the interresidual Ca chemical shift from a one way connectivity spectrum like HN(Co)CA, if the interresidual peak in the HNCA is too weak or overlapping.

        In that case, bigger connectivity cutoffs (cutoffCO, cutoffCA, cutoffCB, and cutoffHA) have to be used due to imperfections in spectrum calibration.

        Nevertheless, Mars does not care where you got the intra- and inter-chemical shifts from!


      3. Be careful of folded peaks!


    For advanced users


      Tensor

        Grid search

          1116 alignment tensor orientations are systematically sampled and for each orientation the deviation D(i,j) between experimental and back-calculated RDCs is determined (equation 1).These 1116 orientations are obtained in a two-step procedure. First, the z-axis of the molecule samples uniformly 122 points on a unit sphere that were determined by a double cubic lattice method (Eisenhaber et al., 1995). Only one quarter of these points have to be taken into account due to the inversion symmetry of RDCs. In a second step, the molecule is rotated around the z-axis in steps of 10? giving a highly uniform and efficient sampling of all possible tensor orientations. All sampled orientations are ranked according to their corresponding D(i,j) values (equation 1) and the lowest D(i,j) value indicates the best estimate for the experimental alignment tensor. Thus, both chemical shifts and RDCs are used, making the extraction of the tensor orientation more robust.


        Exhaustive SVD

          In this method resonances belonging to a specific amino acid type are identified and all possible assignments are searched exhaustively for best-fit of back-calculated to experimental RDCs. The permutation that shows the best agreement identifies the experimental alignment tensor. exhSVD can be applied as long as at least one amino acid type is less than eleven times present in the primary sequence of the protein.


        SVD

          When the assignment is known, an alignment tensor can be obtained by best-fitting experimental dipolar couplings to a 3D structure using singular value decomposition (SVD) (Losonczi et al., 1999). Thus, a two-stage strategy for structure-enhanced assignment can be devised. In the first stage, RDCs are not used and partial assignments are obtained using only chemical shift matching or a combination of chemical shift matching and sequential connectivity information, i.e. using a common backbone assignment strategy. This assignment is assumed to be correct and a alignment tensor is obtained from best-fitting experimental RDCs to the 3D structure. The accuracy of the alignment tensor will depend on the percentage of correct assignments that were obtained in the first assignment phase. Tests show that, even when the percentage of correct assignment is below 50%, the alignment tensor is very close to its correct orientation. This is due to the fact that wrong assignments are not randomly distributed on the primary sequence of the protein. As Ca/Cb chemical shifts depend very much on the type of secondary structure, exchange of assignments mainly takes place between residues located on the same type of regular secondary structure. In addition, b-strands are often part of sheet structures, i.e. different strands are quite collinear. Therefore, residues located in these strands have similar RDCs and back-calculated alignment tensors are not severely affected by an assignment where pseudoresidues are interchanged between two b-strands (data not shown). The applicability of this approach depends mainly on the amount and type of chemical shift information available, i.e. how good is the assignment without RDCs.


        Shape prediction

          Second, molecular alignment tensors of proteins dissolved in nearly neutral bicelles can be predicted from the three-dimensional shape of the protein (Zweckstetter and Bax, 2000). This is particularly attractive as the only information necessary is the 3D structure, i.e. RDCs can be used in the same way as chemical shifts for structure-enhanced assignment. A disadvantage is that this method requires measurement of dipolar couplings in a nearly neutral alignment medium restricting the general applicability of RDC-enhanced assignment. However, recent results indicate that prediction of alignment tensors from 3D structures can be extended to a wide variety of alignment media, when electrostatic interactions are taken into account (Zweckstetter et al., 2003). Thus, this promises to be a valuable approach for obtaining approximate alignment tensors before the start of RDC-enhanced assignment.



    What is MARS ?
    Download
    Setup
    Getting Started
    Setting up input files
    Setting up assignment parameters
    How to run MARS
    Output
    Analysis of assignment / Testing
    Directory structure
    Important points to remember
    For advanced users
Lab home-page at MPIBPC
Lab home-page at DZNE
Home-page DZNE
Home-page MPIBPC

last updated 2014-11-10