Revised July 9, 2007



Examples --- under construction !!

Examples 1 through 10 concern with the 3bp of a hybrid duplex d(AAA):r(UUU). In general, it is easier to go through examples, then learn all the commands from their description. More examples will be added with time. Input files for all examples can be downloaded.

In the examples, commands (keywords) will be shown in boldcase and linked to the appropriate parts of the Command Language section. <file> will stand for a valid unix file name, <data> will stand for numeric data; <integer> - for integer numeric data, etc.


  • Example 1
  • Simple protocol with a single energy call
  • Example 2
  • Energy minimization of one base pair
  • Example 3
  • Energy minimization of the full molecule
  • Example 4
  • Another way to minimize the full molecule
  • Example 5
  • Yet another way to minimize the full molecule
  • Example 6
  • Systematically varying helical twist
  • Example 7
  • A better way to systematically vary helical twist


  • Example 8
  • A protocol for minimization with distance restraints
  • Example 9
  • A Monte Carlo protocol with distance restraints
  • Example 10
  • Another restrained Monte Carlo protocol


  • Example 11
  • A protocol for restrained minimization using cross-relaxation rates
  • Example 12
  • Another minimization protocol with cross-relaxation rates

    Example 1.

    Simple protocol "aaa_1.way": Single energy call for a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid
    
    FOUT
    aaa.end1
    
    INPT
    aaa.inp
    1
    
    STOR
    1
    

    In this protocol, "TITL" sets the title to a character string "hybrid"; "FOUT" sets the output file name to "aaa.end1". "INPT" reads the first record from input file "aaa.inp" (it has one record only, but still the record # "1" must be specified). "STOR" calculates the energy of the current structure and outputs the internal coordinates (into file "aaa.end1"). Ideally, the resulting file "aaa.end1" should be identical to input file "aaa.inp" (except for the date). However, the energy calculations are somewhat platform-dependent (e.g., SGI vs some linux boxes).

    To execute this protocol, type

    miniCarlo -O -s aaa.seq -w aaa_1.way


    Example 2.

    Minimization protocol for energy minimization of the second base pair of a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2). The following four variants produce exactly the same minimization protocols.

    aaa_2a.way:

    TITLE
    hybrid
    
    FOUT
    aaa.end2
    
    INPT
    aaa.inp
    1
    
    MINIM
    100
    PLNE
    2
    
    STOR
    1
    

    aaa_2b.way:

    TITLE
    hybrid
    
    FOUT
    aaa.end2
    
    INPT
    aaa.inp
    1
    
    MINIM
    100
    PAIR
    2
    NUCL
    2
    NUCL
    5
    
    STOR
    1
    

    aaa_2c.way:

    TITLE
    hybrid
    
    FOUT
    aaa.end2
    
    INPT
    aaa.inp
    1
    
    MINIM
    100
    PAIR
    2
    IVAR
    6, 1,2,3,4,5,6
    NUCL
    2
    IVAR
    2, 8,7
    NUCL
    5
    IVAR
    3, 8,7,22
    
    STOR
    1
    

    aaa_2d.way:

    TITLE
    hybrid
    
    FOUT
    aaa.end2
    
    INPT
    aaa.inp
    1
    
    MINIM
    100
    PAIR
    2
    IVAR
    6, 1,2,3,4,5,6
    INCR
      99,99,99,99,99,99
    NUCL
    2
    IVAR
    2, 8,7
    INCR
      99,99
    NUCL
    5
    IVAR
    3, 8,7,22
    INCR
      99,99,99
    
    STOR
    1
    

    This protocol is similar to Example 1, however, before outputting the internal coodinates to file "aaa.end2", 100 cycles of energy minimization is performed: all 11 degrees of freedom associated with PLANE #2 (see Figure 2) are minimized, while all other degrees of freedom are kept fixed. The variant at the left (aaa_2a.way) is the easiest way to select all variables associated with PLANE #2, which consists (in this case) of PAIR #2 and NUCLeotides 2 and 5 (aaa_2b.way). You need to use IVAR (aaa_2c.way) to select individual variables, or to change the order in which they are cycled through in the minimization routine. Parameters ## 1-6 under PAIR are pair parameters (propeller, buckle, opening, shear, stretch, stagger). Parameters 7 and 8 under NUCL are the sugar pseudorotation phase angle and the glycosidic angle, respectively, and 22 is the orientation of the hydroxyl group in NUCLeotide #5 (which is ribo). INCR (in aaa_2d.way) allows one to set the maximum increments for individual parameters during the minimization (which equals 99.0 by default).


    Example 3.

    Minimization protocol "aaa_3.way": Full energy minimization of a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid
    
    FOUT
    aaa.end3
    
    INPT
    aaa.inp
    1
    
    FORN
    2
    
    COMM  minimization of pairs 1,2 and step 1
    MINIM
    300
    PLNE
    1
    STEP
    1
    PLNE
    2
    STOR
    1
    
    COMM  minimization of pairs 2,3 and step 2
    MINIM
    300
    PLNE
    2
    STEP
    2
    PLNE
    3
    STOR
    1
    
    NEXT
    
    MMOL
    aaa_end3.pdb
    
    This is a more complex minimization protocol. It does complete minimization of this molecule, which is split into two parts: (1) 300 cycles of minimization of planes 1 and 2 and step 1, and (2) 300 cycles minimization of planes 2 and 3 and step 2. The whole process is repeated two times (loop FORN -- 2 .... NEXT ). In the end, a pdb file "aaa_end3.pdb" is written. Try to experiment, changing number of cycles of minimization, and number of times the whole process is repeated (FORN). Note that in the end the output file with internal coordinates "aaa.end3" will have four records created by STOR statements: two in each FORN cycle; the last record corresponds to the minimized structure.


    Example 4.

    Minimization protocol "aaa_4.way": Another way to minimize the DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid
    FOUT
    aaa.end4
    INPT
    aaa.inp
    1
    FORN
    2
      OFST
      -1
      FORN
      2
        ADDD
        OFST
        1
        COMM  minimization of pairs 1,2 and step 1
        MINIM
        28, 300
        PLNE
        1
        STEP
        1
        PLNE
        2
      NEXT
    NEXT
    STOR
    1
    MMOL
    aaa_end4.pdb
    
    This protocol produces the same result as in Example 3, but here, the use of loops and OFST is illustrated. The inner FORN ... NEXT loop is executed twice: first time with OFST = 0, and second time with OFST = 1. Correspondingly, first time, planes 1, 2 and step 1 are minimized, and second time, planes 2, 3, and step 2 are minimized. STOR statement is placed outside of loops here, so that "aaa.end4" will have only one record, identical to the last record of "aaa.end3".
    Note the indentation in this protocol, which is optional.


    Example 5.

    Minimization protocol "aaa_5.way": Yet another way to minimize the DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid
    FOUT
    aaa.end5
    INPT
    aaa.inp
    1
    FORN
    2
      OFST
      -1
      FORN
      2
        ADDD
        OFST
        1
        FILE
        min.way
      NEXT
    NEXT
    STOR
    1
    MMOL
    aaa_end5.pdb
    
    The protocol in this example will produce the same result as in Examples 3 and 4, if the following file with the name "min.way" is present in the working directory:
    COMM  minimization of pairs 1,2 and step 1
    MINIM
    28, 300
    PLNE
    1
    STEP
    1
    PLNE
    2
    


    Example 6.

    Minimization protocol "aaa_6.way": Systematically varying helical twist in a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    COMM "scanning" twist-1
    TITLE
    hybrid
    FOUT
    aaa.end6
    INPT
    aaa.end4
    1
    CHNG
    STEP
    1, 1, 1, 30
    FORN Varying Twist of Step 1 from 31 to 40 deg.
    10
      ADDD
      STEP
      1, 1, 1,  1.0
      COMM  minimization of pairs 1,2 and step 1
      COMM    except Twist in STEP 1
      MINIM
      300
      PLNE
      1
      STEP
      1
      IVAR note that parameter #1 (twist) is excluded from the list
      5, 2,3,4,5,6
      PLNE
      2
      STOR
      1
    NEXT
    
    This example shows how to vary systematically helical parameters. This protocol calculates a series of 10 structures with twist parameter in the first step varied from 31 to 40 degrees. It starts with the structure "aaa.end4" previously minimized in Example 4. Then, using CHNG, it changes the twist parameter (#1) in the first step to 30 degrees. After that, the FORN ... NEXT loop is cycled 10 times; within each iteration, the twist value is increased by 1 degree, the structure is energy minimized, and the result is stored in the file "aaa.end6".

    Note that keyword STEP is used in two different contexts here. First, it is used under CHNG to select, in STEP #1, one parameter (#1, i.e., Twist) and to change it to 30 degrees. Then, it is used under MINI to select five parameters ## 2,3,4,5,6 (i.e., Tilt, Roll, Shift, Slide, and Rise) -- all parameters except for the Twist (#1), which is excluded from the minimization.

    Also note that commentaries may be added in the protocol in the end of any line after the keyword.

    The shortcoming of this particular protocol is a relatively big jump in the twist #1 in the beginning of the protocol. This parameter is about 35.9 degrees after the execution of protocol "aaa_4.way" of Example 4 (please check it). When this parameter is changed to 31 degrees later in this protocol, the structure changed too much, and the following energy minimization may not find the global minimum with twist #1 = 31. The following example shows how to deal with this problem.


    Example 7.

    Minimization protocol "aaa_7.way": A better way to systematically vary helical twist in a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    COMM "scanning" twist-1
    TITLE
    hybrid
    FOUT
    junk.end
    INPT
    aaa.end4
    1
    CHNG
    STEP
    1, 1, 1, 36
    FORN Gradually changing Twist of Step 1 down to 31deg.
    5
      ADDD
      STEP
      1, 1, 1,  -1.0
      MINIM
      300
      PLNE
      1
      STEP
      1
      IVAR
      5, 2,3,4,5,6
      PLNE
      2
      STOR
      1
    NEXT
    
    FOUT new output file
    aaa.end7
    CHNG
    STEP
    1, 1, 1, 30
    FORN Varying Twist of Step 1 from 31 to 40 deg.
    10
      ADDD
      STEP
      1, 1, 1,  1.0
      MINIM
      300
      PLNE
      1
      STEP
      1
      IVAR
      5, 2,3,4,5,6
      PLNE
      2
      STOR
      1
    NEXT
    
    Here, the twist #1 is first gradually changed to 31 deg. (instead of changing it in one step in Example 6). The STOR output is written in file "junk.end". After that, the output file is changed to "aaa.end7", and twist #1 is systematically changed from 31 to 40 degrees. Compare the results (aaa.end7) with those of the previous example (aaa.end6).


    Example 8.

    Refinement protocol "refine_1.way": Restrained minimization protocol for a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid restrained minimization
    FOUT
    aaa_rmin.1
    INPT
    aaa.end4
    1
    REST
    Aform.restraints
    WRES
    0.5
    STOR
    1
    ROUT
    1
    FORN -- increasing WRES
    6
      MULT
      WRES
      2.0
      FILE
      min_3bp.way
      STOR
      1
      ROUT
      1
    NEXT
    
    WRES -- relaxing WRES
    0.5
    FILE
    min_3bp.way
    FOUT
    aaa_rmin.2
    STOR
    1
    ROUT
    2
    MMOL
    aaa_rmin_2.pdb
    
    This protocol starts with the input of the last record of "aaa.end4", which is an energy-minimized structure of the hybrid d(AAA):r(UUU) calculated in Example 4). This structure is a typical B-conformation. Then, command REST inputs distance restraints from the file "Aform.restraints" (see description of format of this file). The restraints were generated for an energy-minimized A-conformation for this molecule; force constants were set to 10 kcal/(mol·Å2) in this file. Note that there are no "holonomic" (meaning "fake") H-bond restraints for the base pairs in "Aform.restraints", because during minimization base pairs are not going to be broken.

    The weight of distance restraints WRES is reduced to 0.5; the initial structure is output to the file "aaa_rmin.1" (using STOR) and initial distance deviation is output to the same file with ROUT 1 (the initial average distance deviation is a high value of 0.95 Å).

    The structure is refined in six iterations of restrained minimization (FORN 6 ... NEXT), during which the weight of distance restraints is exponentially increased (using multiplication of the scale factor WRES by 2.0 in each iteration). A sub-protocol specified in the file "min_3bp.way" is used for the minimization:
    COMM this protocol minimizes 3 bp
    FORN
    2
    OFST
    -1
    FORN
    2
      ADDD
      OFST
      1
      COMM minimization of pairs 1,2 and step 1
      MINI
      300
      PLNE
      1
      STEP
      1
      PLNE
      2
    NEXT
    OFST
    0
    
    NEXT
    
    This sub-protocol is essentially identical to the one used in Example 4. In the end of each iteration, internal coordinates and distance deviations are output in the file "aaa_rmin.1". After the six iterations, the structure is already converted in the A-conformation (check it!). (Please note that such a B-to-A convertion cannot be accomplished with simple restrained minimization using Cartezian coordinates- based methods; this normally requires simulated annealing using restrained molecular dynamics).

    In the second half of the protocol, the weight of distance restraints is reduced and the structure is again restrained minimized; this improves its conformational energy without compromising strongly the restraint energy (because the structure has already converted to an energy minimum corresponding to the A form). The final result is output into the file "aaa_rmin.2" (STOR), and distance deviations and all individual distance restraints are output in the same file (ROUT 2). The final average distance deviation is 0.06 Å; the maximum individual distance deviation us 0.34 Å (for the H8 3 - 2H2' 3 restraint).

    Note that the file "aaa_rmin.2" has only one record with internal coordinates (helical parameters) and all individual distance restraints (output with ROUT 2). File "aaa_rmin.1" with intermediate helical parameters has seven records, each of them also contains the summary of distance restraints (output with ROUT 1).


    Example 9.

    Refinement protocol "refine_2.way": Restrained Monte Carlo protocol for a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    TITLE
    hybrid restrained Monte Carlo
    FOUT
    aaa_rmc.end1
    INPT
    aaa.end4
    1
    REST
    Aform_hb.restraints
    WRES
    0.5
    STOR
    1
    ROUT
    1
    
    INCS
    1.5
    FORN -- increasing WRES
    8
      MULT
      WRES
      2.0
      MULT
      TEMP
      1.25
      FORN -- 4,000 iterations (20 x 200)
      20
        NBLS
        FORN
        200
          FILE
          3bp.shak
        NEXT
        OUTP
        0
        OUTP
        10
      NEXT
      STOR
      1
      ROUT
      1
    NEXT
    
    WRES -- relaxing WRES
    0.5
    FILE
    min_3bp.way
    FOUT
    aaa_rmc.end2
    STOR
    1
    ROUT
    2
    
    Protocol of this example accomplish the same goal as the protocol of Example 8, i.e., it refines A-conformation starting with initial B-form. However, this protocol performs restrained Metropolis Monte Carlo simulations instead of restrained minimization. It uses a set of distance restraints "Aform_hb.restraints", which has fake Hbond restraints. The reason for this is to prevent the strands from separating when the temperature is increased during the protocol; these Hbonds can be excluded during the final restrained minimization.

    The weight of restraints WRES is increased in four eight; simultaneously is increased the temperature of the simulation. A file with the minimization sub-protocol "min_3bp.way" in Example 8 is replaced here with the sub-protocol "3bp.shak", which describes one iteration of Monte Carlo. Each iteration is repeated 4,000 times (20 x 200) after which the current structure is stored in "aaa_rmc.end1". After each 200 iterations, a short info is written onto stdio (OUTP 0) and into file "info_current." (OUTP 10). Also, the list of non-bonded interactions is updated every 200 iterations (NBLS).

    After 4,000 iterations, the staructure is already converted into A-conformation (check the last record of file "aaa_rmc.end1"). In the end, the structure is additionally restrained- minimized with reduced WRES; this serves to improve the conformational energy of the structure. The resulting structure is stored in file "aaa_rmc.end2".

    Below is shown file "3bp.shak" which has a sub-protocol describing a single iteration of Monte Carlo:
    COMM one iteraction of Monte Carlo
    OFST
    -1
    FORN
    2
      ADDD
      OFST
      1
      SHAK
      PLNE
      1
      SHAK
      STEP
      1
      SHAK
      PLNE
      2
    NEXT
    OFST
    0
    ITER
    
    In this sub-protocol, a single Monte Carlo iteration (set by ITER) consists of six elementary Metropolis trials (SHAK). In the first cycle of the FORN ... NEXT loop, OFST is zero, and plane #1, step #1, and plane #2 are shaked. In the second cycle of the loop, OFST is 1, and plane #2, step #2, and plane #3 are shaked. There is a flexibility in how the set of internal coordinates is split among different SHAKEs. It is possible to shake more than one step or one pair at a time, or it is possible to shake even smaller sets of variables at a time than shown in this example. However, it is generally advantageous to combine together variables that are likely to be correlated. The default values for the maximum increments in each shake are normally used; they can be specified explicitly with INCR after the explicit selection of variables with IVAR, similarly to protocol "aaa_2d.way" from Example 2. Alternatively, the default maximum increments can be scaled with INCS, as shown in this example. The maximum increments for the single shake must be adjusted to obtain the desired rejection-to-acceptance ratio. The ratio of rejected conformations is reported on screen during OUTP 0, and in the file "info_current." during OUTP 10. Note that this ratio also depends on the temperature and on distance restraints used. Increasing the maximum increments leads to the increase of the ratio of rejected conformations, because large random perturbations likely lead to a large increase in energy. Generally, it is considered that the rejection-to-acceptance ratio must be ca. 0.5 as a compromise between very slow changes in conformation and high rejection ratio. However, in my experience, during restrained Monte Carlo, a somewhat increased rejection-to-acceptance ratio makes the covergence faster.

    Note that it is not necessary to set OFST to zero in the very end of this sub-protocol, but it is safer to return it to its default value; then this file ("3bp.shak") can be used in many different situations.


    Example 10.

    Refinement protocol "refine_3.way": Another restrained Monte Carlo protocol for a DNA:RNA hybrid d(AAA):r(UUU) (Figure 2).

    COMM -- first part --
    TITLE
    hybrid restrained Monte Carlo
    FOUT
    aaa_rmc.end3
    INPT
    aaa.end4
    1
    REST
    Aform_hb.restraints
    WRES
    0.5
    STOR
    1
    ROUT
    1
    FORN -- increasing WRES
    8
      MULT
      WRES
      2.0
      MULT
      TEMP
      1.25
      FORN -- 4,000 iterations (20 x 200)
      20
        NBLS
        FORN
        200
          FILE
          3bp.shak
        NEXT
        OUTP
        0
        OUTP
        10
      NEXT
      STOR
      1
      ROUT
      1
    NEXT
    
    COMM -- second part --
    RSAV -- restarting Monte Carlo chain
    
    WRES -- relaxing WRES
    0.5
    TEMP
    100
    
    FORN low-temp 2,000 iterations
    10
      NBLS
      FORN
      200
        FILE
        3bp.shak
      NEXT
      OUTP
      0
      OUTP
      10
    NEXT
    
    FOUT
    aaa_rmc.end4
    STOR
    1
    ROUT
    1
    
    FAVE
    aaa_rmc.out4
    AVER
    
    LDAV -- load the averages
    STOR
    1
    ROUT
    1
    
    FILE
    min_3bp.way
    STOR
    1
    ROUT
    2
    
    The purpose of this example is to illustrate the usage of commands RSAV and LDAV. The first part of this protocol is identical to that of Example 9, so that the first nine records of "aaa_rmc.end3" must be identical to "aaa_rmc.end1".

    The second part of this protocol starts with restarting the Monte Carlo chain (RSAV); it zeroes the iteration count and all running sums previously accumulated using ITER command. Then it runs another 2,000 iterations at 100K. After that it averages accumulated sums and loads averages into the current structure with LDAV. And finally, the average structure is further restrained minimized ("min_3bp.way").

    Compare the usage of commands AVER and LDAV in this protocol: AVER averages the accumulated sums and writes mean and std values in the file aaa_rmc.out4 (set by FAVE). LDAV also averages the accumulated sums but it does not make any output; instead it substitutes the current internal coordinates with the averages. AVER and LDAV can be used independent of each other. The averaged internal coordinates were immediately stored in "aaa_rmc.end3" with STOR, and then stored again after 2,000 iterations at 100K.


    Example 11.

    A protocol for multiple-copy restrained minimization using cross-relaxation rates (file pqx1.way). This protocol must be invoked using command
    miniCarlo -m 2 -O -s aaa.seq -w pqx1.way
    
    TITL
    rate-based restrained minimization
    INPT
    aaa.end4
    1
    FOUT
    aaa_pdq.end1
    
    PDQX
    0
    ab70.spt
    
    WOBJ
    5.0
    
    FORN
    4
      MULT
      WOBJ
      2.0
      COMM minimization of 3 bp in 2 steps
      FORN
      2
        OFST
        -1
        FORN
        2
          ADDD
          OFST
          1
          COMM  minimization of pairs 1,2 and step 1
          MINI
          300
          PLNE
          1
          STEP
          1
          PLNE
          2
        NEXT
        OFST
        0
      NEXT
      STOR
      1
    NEXT
    XMOL
    aaa_pdq_end1.pdb
    

    This protocol is similar to the protocol of Example 8, however cross-relaxation rates (file "ab70.spt") are used instead of distance restraints, and the program miniCarlo is run with two copies of the hybrid d(AAA):r(UUU) (option "-m 2"). It starts with the input of the last record of "aaa.end4", which was energy- minimized (unrestrained) in Example 4. Because miniCarlo is run with two copies in this example, and #4 is the last record in file "aaa.end4", INPT inputs two identical conformations (this is a typical B form).

    "Experimental" cross-relaxation rates for this example (specified in file "ab70.spt") were prepared for an ensemble of A form (70%) and B form (30%) of the hybrid d(AAA):r(UUU) using the program "corma. The structures used for the corma simulation ("aform.end" and "bform.end") can be downloaded with this example to compare with the results of this protocol.

    The spt-file with experimental cross-relaxation rates is input with the PDQX command. The PDQX mode is set to zero, which means that copies' probabilities will be calculated during each energy call (this is the most useful value of this parameter). The relaxation rates-based objective function Qr will be added as penalty to the total energy of each copy. The initial weight of Qr is set to 5.0 with WOBJ (the default value of this parameter is 1.0).

    The structures (both copies) are refined in four iterations of minimization (FORN 4 ... NEXT) during which the weight of Qr is exponentially increased. (In contrast to Example 8, the minimization commands are not called from the file "min_3bp.way", but they are arranged in-line in the main protocol "pqx1.way"). Each command MINI is executed sequentially on two copies (note a slightly changed screen output when minimization is performed on multiple copies).

    Note that each STOR command outputs helical parameters for each copy sequentially, so that there are eight records in the file "aaa_pdq.end1" after the protocol is completed (records ## 7 and 8 correspond to the final conformations of copies 1 and 2, with probabilities of 0.77 and 0.23, respectively). Command XMOL creates pdb files corresponding to these two conformations, with the file names "aaa_pdq_end1.pdb_copy__1" and "aaa_pdq_end1.pdb_copy__2".

    The resulting ensemble of two conformers has a quite low objective function Qr of 0.186. Nevertheless, while the second copy is a B-type conformation, the first copy was not converted completely into the A-form: residue U6 still has a S-type sugar pucker. Further increasing weight WOBJ does not help (try to experiment with this protocol): apparently, the ensemble of two conformers was trapped in a stable local (incorrect) minimum not very far from the global (correct) minimum (compare this solution with the target structures, "aform.end" and "bform.end"). One probable reason for this is that the three base pairs of the hybrid were minimized in two overlapping fragments (FORN 2 ... NEXT): first pairs ## 1 and 2 and step # 1 (with OFST 0), and then pairs ## 2 and 3 and step # 2 (with OFST 1). The protocol of Example 12 is more successful in finding the target conformations.


    Example 12.

    Another protocol for restrained minimization using cross-relaxation rates (file pqx2.way). This protocol must be invoked using command
    miniCarlo -m 2 -O -s aaa.seq -w pqx2.way
    
    TITL
    rate-based restrained minimization
    INPT
    aaa.end4
    1
    FOUT
    aaa_pdq.end2
    
    PDQX
    0
    ab70.spt
    
    WOBJ
    5.0
    
    FORN
    10
      MULT
      WOBJ
      2.0
      FORN
      2
        COMM minimization of pairs 1,2,3 and steps 1,2
        MINI
        700
        PLNE
        1
        STEP
        1
        PLNE
        2
        STEP
        2
        PLNE
        3
      NEXT
      STOR
      1
    NEXT
    XMOL
    aaa_pdq_end2.pdb
    

    This protocol is very similar to the protocol of Example 11 (try to find all the differences), however, this protocol finds successfully the ensemble of target structures ("aform.end" and "bform.end") with the correct probabilities and a very low Qr of 0.0005.