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	MR Tutorial Bath - Pepsin case study

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4. Case study 1 - hexagonal pepsin

Target:

Hexagonal pepsin (point group 622, 1 molecule/asymmetric unit).

Model:

Penicillopepsin (PDB code 3APP).

4.1. Rotation function (AMORE / ROTFUN) with F's

4.1.1. AMoRe SORTFUN

Convert the reflection data file into the internal format used by the AMORE program.

#
amore  HKLIN hexpep  HKLPCK0 hexpep.hkl  <<EOD
TITLE  ** packing h k l F for crystal **
SORT
LABIN  FP=FOHEXPEP  SIGFP=SIGHEXPEP
EOD

4.1.2. AMoRe TABFUN

Compute a molecular Fourier transform for use in structure factor calculation by interpolation.

#
amore  XYZIN1 penpep  XYZOUT1 penpep_model  table1 penpep.tab  <<EOD
TITLE  Produce table for penicillopepsin model.
TABFUN NOROTA                 ! No rotate for comparison.
CRYST  CELL 67.4 67.4 290.1 90 90 120
MODEL  1  BREP 10
SAMPLE 1  RESO 2.8            ! Beyond native resolution for interpolation.
EOD

Note that the steps in the procedure need not be run as separate jobs as illustrated in the two previous examples; this has only been done in order to simplify explanation of the individual steps. The next example shows several steps being run sequentially in a single job.

4.1.3. AMoRe ROTFUN GENERATE

Compute structure factor amplitudes for the search model in space group P1 in a large rectangular cell.

4.1.4. AMoRe ROTFUN CLMN

Compute spherical harmonic coefficients for the target and model Pattersons.

4.1.5. AMoRe ROTFUN CROSS

Compute the cross-RF.

#
amore  HKLPCK0 hexpep.hkl  table1 penpep.tab  HKLPCK1 penpep.hkl  \
       CLMN0 hexpep.clmn  CLMN1 penpep.clmn  MAPOUT hexpep_rotfun  <<EOD
ROTFUN
TITLE  Generate HKLPCK1 from penicillopepsin model.
GENER  1  RESO 20 3  CELL 83 82 92
CLMN   CRYST  RESO 20 3  SHARP -10  SPHERE 30
CLMN   MODEL 1  RESO 20 3  SHARP -10  SPHERE 30
ROTATE CROSS  MODEL 1  BMAX 90  PKLIM .25  NPIC 20          ! Beta = 0-90
EOD
\rm hexpep_rotfun.map

Peak lists from ROTFUN (correct solution is highlighted)

Radius = 25Å. B = -10Ų.

Radius = 30Å. B = 0.

Radius = 30Å. B = -10Ų.

Rotation function of hexagonal pepsin (F's).

The table below shows values of the signal/noise ratio DS/s for the correct peak relative to the highest background peak. A "-" indicates that the correct peak was not present in the top 20 peak list.

Note: Other combinations of resolution limits (8-3Å, 20-3.5Å, 8-3.5Å), radius or B parameter did not give correct peak in list.

4.2 Rotation function (AMORE / ROTFUN) with E's

4.2.1. PDBSET

Modify the PDB header in the coordinate file.

#
pdbset  XYZIN penpep  XYZOUT penpep_rfcell <<EOD
SPACEG P1
CELL   83 82 92
EOD

4.2.2. SFALL

Compute structure factor amplitudes in space group P1 for the search model in a large rectangular cell.

#
# Structure factors for penicillopepsin model in RF cell.
#
sfall  XYZIN penpep_rfcell  HKLOUT penpep_rfcell <<EOD
MODE   SFCALC XYZIN
SFSG   1
SYMM   1
RESO   20 3
BADD   10
LABOUT PHIC=PC
EOD

4.2.3. ECALC

Normalise the observed and calculated amplitudes.

#
ecalc  HKLIN hexpep  HKLOUT hexpep_ecalc  <<EOD
TITLE  ** Ecalc for target crystal**
LABIN  FP=FOHEXPEP  SIGFP=SIGHEXPEP
EOD

ecalc  HKLIN penpep_rfcell  HKLOUT penpep_ecalc  <<EOD
TITLE  ** Ecalc for model**
LABIN  FP=FC
EOD

4.2.4. AMoRe SORTFUN

Convert the normalised observed and calculated reflection data files into the internal format used by the AMORE program.

#
amore  HKLIN hexpep_ecalc  HKLPCK0 hexpep_ecalc.hkl  <<EOD
TITLE  ** packing h k l E for target crystal **
SORT
LABIN  FP=E  SIGFP=E
EOD

amore  HKLIN penpep_ecalc  HKLPCK0 penpep_ecalc.hkl  <<EOD
TITLE  ** packing h k l E for model **
SORT
LABIN  FP=E  SIGFP=E
EOD

4.2.5. AMoRe ROTFUN CLMN

Compute spherical harmonic coefficients for the target and model Pattersons.

4.2.6. AMoRe ROTFUN CROSS

Compute the cross-RF.

#
amore  HKLPCK0 hexpep_ecalc.hkl  HKLPCK1 penpep_ecalc.hkl  \
       CLMN0 hexpep_ecalc.clmn  CLMN1 penpep_ecalc.clmn  \
       MAPOUT hexpep_ecalc_rotfun  <<EOD
ROTFUN
TITLE  Rotation function with E's..
CLMN   CRYST  RESO 20 3  SPHERE 25
CLMN   MODEL 1  RESO 20 3  SPHERE 25
ROTATE CROSS  MODEL 1  BMAX 90  PKLIM .25  NPIC 20
EOD
\rm hexpep_ecalc_rotfun.map

Peak list from ROTFUN.

Note that 2 peaks separated by a small rotation angle (about 14^o) are observed in the Rotation function based on E's, whereas only a single peak was observed when F's were used; thus normalisation produces a Rotation function map with a much higher effective resolution. The splitting is due to hinge-bending giving differing orientations of the N- and C-terminal domains of the aspartic proteinase. In hindsight it might have been better to separate the model into its constituent domains, and treat it as a 2-molecule problem.

Rotation function of hexagonal pepsin (E's).

The table below shows values of DS/s.

The table below shows the effect on the signal/noise ratio of the RF of omitting the largest intensities (resolution limits: 20-2Å).

Note: Negative signal/noise means that the correct peak is lost in the noise!

4.3. Translation function (AMORE / TRAFUN) in space group P6₅22 with F's

4.3.1. AMoRe TRAFUN

For the best RF solutions, compute the TF's.

#
amore  HKLPCK0 hexpep.hkl  table1 penpep.tab  MAPOUT hexpep_trafun.map <<EOD
TRAFUN CB  RESO 20 3.5
TITLE  Translation function
SYMM   P6522
SOLUTIONRC   1    30.14    37.23   256.28  0.00000  0.00000  0.00000  5.6  0.0
SOLUTIONRC   1    35.25    30.37   244.50  0.00000  0.00000  0.00000  5.5  0.0
SOLUTIONRC   1    41.00    28.32    19.00  0.00000  0.00000  0.00000  5.4  0.0
SOLUTIONRC   1    32.75    85.70   129.88  0.00000  0.00000  0.00000  5.0  0.0
SOLUTIONRC   1    32.99    78.33    35.83  0.00000  0.00000  0.00000  4.9  0.0
EOD

Translation function of hexagonal pepsin (F's).

The table below is for the correct RF solution.

4.3.2. AMoRe TRAFUN 2

No NCS, so this step not applicable.

4.3.3. AMoRe FITFUN

For the best TF solutions, do rigid-body refinements and choose the solution with the highest correlation coefficient.

#
amore  HKLPCK0 hexpep.hkl  table1 penpep.tab  <<EOD
FITFUN RESO 20 3.5
TITLE  Rigid body refinement
SYMM   P6522
REFSOL AL BE GA X Y Z BF
SOLUT_1     1   43.50   21.00   14.00  0.0772  0.6903  0.2988 19.5 53.0   1
SOLUT_1     1   43.50   21.00   14.00  0.5903  0.1203  0.3246 13.3 54.4   2
SOLUT_1     1   43.50   21.00   14.00  0.8148  0.4523  0.3953 12.7 54.9   9
SOLUT_2     1   42.17   29.66   26.30  0.0719  0.2201  0.3324 13.4 54.7   4
SOLUT_2     1   42.17   29.66   26.30  0.0789  0.2299  0.4226 13.1 54.9   2
SOLUT_3     1   52.38   54.25  128.35  0.6740  0.9740  0.4168 13.9 54.9   9
SOLUT_3     1   52.38   54.25  128.35  0.4894  0.9219  0.2713 12.9 55.4   2
SOLUT_4     1   32.72   58.97   66.17  0.0550  0.0375  0.0206 12.9 55.1   1
SOLUT_5     1   38.50   59.00   65.69  0.9230  0.1232  0.2007 13.5 53.8   1
SOLUT_5     1   38.50   59.00   65.69  0.9197  0.1238  0.0890 12.5 54.8   2
EOD

Sample output from AMORE / FITFUN.

 INITIAL POSITIONS
    1    43.500    21.000    14.000  0.077200  0.690300  0.298800     19.500  53.000
 FINAL POSITIONS
 SOLUTIONF   1    43.58    22.67    15.07  0.07790  0.69162  0.29897 26.6 51.3

 INITIAL POSITIONS
    1    43.500    21.000    14.000  0.590300  0.120300  0.324600     13.900  54.900
 FINAL POSITIONS
 SOLUTIONF   1    45.54    21.02    12.42  0.59161  0.12234  0.32435 18.3 54.4

 INITIAL POSITIONS
    1    43.500    21.000    14.000  0.814800  0.452300  0.395300     13.500  53.800
 FINAL POSITIONS
 SOLUTIONF   1    42.93    21.40    14.83  0.81292  0.44990  0.39566 18.3 53.3

Rigid-body refinement of hexagonal pepsin.

4.3.4. PDBSET

Apply the optimised rotation matrix and translation vector to the model coordinates output by AMORE / TABFUN.

#
pdbset XYZIN penpep_model  XYZOUT hexpep  <<EOD
SPACEG P6522
CELL   67.4 67.4 290.1 90 90 120
ROTATE EULER 42.68 26.21 19.27
SHIFT  FRACT  0.07790  0.69162  0.29897
EOD

4.4 Translation function (TFFC / FFT) in space group P6₅22 with E's

4.4.1. PDBSET

Apply the appropriate rotation matrix, calculated from the Eulerian angles of the peak(s) in the RF, to the model coordinates. Also modify the PDB header in the coordinate file.

#
pdbset XYZIN penpep  XYZOUT penpep_tfcell  <<EOD
SPACEG P1
CELL   67.4 67.4 290.1 90 90 120
ROTATE EULER 42.68 26.21 19.27
EOD

4.4.2. SFALL

Calculate structure factor amplitudes and phases.

#
# Structure factors for penicillopepsin model in TF cell.
sfall  XYZIN penpep_tfcell  HKLOUT penpep_tfcell <<EOD
MODE   SFCALC XYZIN
SFSG   1
SYMM   1
RESO   20 3
BADD   10
LABOUT PHIC=PC
EOD

4.4.3. CAD

Combine the columns for the observed reflection data with those for the calculated molecule into a single file.

#
cad    HKLIN1 hexpep  HKLIN2 penpep_tfcell  HKLOUT hexpep_cad  <<EOD
RESO   OVER 20 3
TITLE  COMBINING  hexagonal pepsin Fobs & penicillopepsin Fcalc.
LABIN  FILE 1  E1=FOHEXPEP E2=SIGHEXPEP
CTYPE  FILE 1  E1=F        E2=Q
LABIN  FILE 2  E1=FC       E2=PC
CTYPE  FILE 2  E1=U        E2=V
LABOUT FILE 2  E1=FC       E2=PC
END
EOD

4.4.4. TFFC

Compute the Fourier coefficients for the Translation function.

#
tffc   HKLIN hexpep_cad  HKLOUT hexpep_tffc  <<EOD
TITLE  tffc on hexagonal pepsin
SPACEG P6522
RESO   20 3.5
LABIN  FP=FOHEXPEP SIGFP=SIGHEXPEP
EOD

4.4.5. FFT

Compute the Fourier transform to get the Translation function map.

#
fft    HKLIN hexpep_tffc  MAPOUT hexpep_tffc  <<EOD
TITLE  FFT of tffc map
XYZLIM 0 1  0 1  0 .5               ! Limits for point group 622
GRID   128 128 512
LABIN  A=A  B=B
EOD

4.4.6. MAPSIG

Search the Translation function map for peaks; one peak should stand out from the rest with relative signal/noise > 1.

#
mapsig MAPIN hexpep_tffc  PEAK_LIST hexpep_tffc.pkl  <<EOD
EOD
\rm hexpep_tffc.map

Peak list from TFFC.

It may be necessary to try an alternative space group (in this case P6₁22):

#
tffc   HKLIN hexpep_cad  HKLOUT hexpep_tffc  <<EOD
TITLE  tffc on hexagonal pepsin in alternative space group.
SPACEG P6122
RESO   20 3.5
LABIN  FP=FOHEXPEP SIGFP=SIGHEXPEP
EOD

if ($status) exit

fft    HKLIN hexpep_tffc  MAPOUT hexpep_tffc  <<EOD
TITLE  FFT of tffc map
XYZLIM 0 1  0 1  0 .5
GRID   128 128 512
LABIN  A=A  B=B
EOD

if ($status) exit

mapsig MAPIN hexpep_tffc  PEAK_LIST hexpep_tffc.pkl  <<EOD
EOD
\rm hexpep_tffc.map

Peak list from TFFC.

Translation function of hexagonal pepsin (E's).

ª Alternative space group P6₁22.

4.4.7. TFFC, FFT, MAPSIG

No NCS, so this step not applicable.

4.4.8. PDBSET

Apply the translation vector to the rotated model coordinates.

#
pdbset XYZIN penpep_tfcell  XYZOUT hexpep  <<EOD
SPACEG P6522
SHIFT  FRACT  .968 .268 .264
EOD

4.5 Rotation function (X-PLOR)

The X-PLOR program can also be used to compute the cross-RF and do rigid or segmented body refinement in Patterson space (Patterson Correlation or PC refinement). In theory this should be particularly useful in cases where there is limited flexibility such as hinge-bending. This is common in multi-domain proteins, including the aspartic proteinases.

4.5.1. If necessary, convert reflection file to X-PLOR format

#
mtz2various  HKLIN hexpep  HKLOUT hexpep.xpl  <<EOD
OUTPUT XPLOR
LABIN  FP=FOHEXPEP  SIGFP=SIGHEXPEP
EOD

4.5.2. Create the X-PLOR structure file

#
xplor  << \EOD
 remarks  file  generate/generate.inp
 remarks  Generate structure file and hydrogens for a protein
 topology
   @topPAR:tophcsdx.pro                        { Read topology file }
 end
 parameter
   @topPAR:parhcsdx.pro                      { Read empirical potential }
                                             { parameter file CHARMM19 }
                                             { with modifications }
   nbonds                                    { This statement specifies the}
      atom cdie shift eps=1.0  e14fac=0.4    { nonbonded interaction energy}
      cutnb=7.5 ctonnb=6.0 ctofnb=6.5        { options.  Note the reduced }
      nbxmod="5" vswitch                       { nonbonding cutoff to save }
   end                                       { some CPU time. This statement}
                                             { overwrites the defaults in the}
                                             { parameter file. }
 end
 segment                                     { Generate protein }
   name="    "                               { This name has to match the }
                                             { four characters in columns 73}
                                             { through 76 in the coordinate}
                                             { file, in XPLOR this name is }
                                             { name is referred to as SEGId.}
   chain
     @topPAR:toph19.pep                      { Read peptide bond file }
     coordinates @penpep.brk end             { interpret coordinate file to}
   end                                       { obtain the sequence }
 end
                                             { Sometimes different atom }
 vector do (name="O") ( name OT1 )           { names are used... }
 vector do (name="OT") ( name OT2 )
 vector do (name="CD1") ( name CD and resname ile )
 coordinates @penpep.brk end                 { Here we actually read the }
                                             { coordinates }
 vector do ( B=10 ) ( all )                  { B's are zero: reset all to 10}
 flags exclude vdw elec end                  { Do QUICK hydrogen building w/o}
                                             { vdw and elec terms }
 hbuild                                      { This statement builds }
    selection=( hydrogen )                   { missing hydrogens which are }
    phistep="4"                                { needed for the force field }
 end
 write coordinates output=penpep_xplor.brk end      { Write out coordinates}
 write structure output=penpep.psf end       { Write out structure file }
 stop
\EOD

4.5.3. Compute the Rotation function and search for peaks

#
xplor  << \EOD
 remarks file xtalmr/rotation.inp  -- cross rotation function
 remarks                              (model P1 vs crystal)
{===>} structure @penpep.psf end                        { read structure file}
{===>} coor @penpep_xplor.brk                           { read coordinates }
{===>}                              { specify location of Patterson map files}
evaluate ( $p1_map="p1.map" )
evaluate ( $p2_map="p2.map" )
{===>}
evaluate ( $max_vector=30 )        { maximum Patterson vector to be searched}
evaluate ( $m_max_vector=-$max_vector )
xrefin                             { make Patterson P1 map of model in P1 box}
{===>}                           { the P1 box has to be larger than twice the}
                                 { the extend of the model in each direction}
   a=100.0 b=100.0 c=120.0 alpha=90.0 beta=90.0 gamma=90.0
   symmetry=(x,y,z)
   SCATter ( chemical C* )
   2.31000 20.8439 1.02000 10.2075 1.58860 .568700 .865000 51.6512 .215600
   SCATter ( chemical N* )
   12.2126 .005700 3.13220 9.89330 2.01250 28.9975 1.16630 .582600 -11.529
   SCATter ( chemical O* )
   3.04850 13.2771 2.28680 5.70110 1.54630 .323900 .867000 32.9089 .250800
   SCATter ( chemical S* )
   6.90530 1.46790 5.20340 22.2151 1.43790 .253600 1.58630 56.1720 .866900
   SCATter ( chemical P* )
   6.43450 1.90670 4.17910 27.1570 1.78000 0.52600 1.49080 68.1645 1.11490
   SCATter ( chemical FE* )
   11.1764 4.61470 7.38630 0.30050 3.39480 11.6729 0.07240 38.5566 0.97070
{===>}          { allocate sufficient space for the reflections of the P1 box}
   nreflections=200000
{===>}
   resolution 20 3                          { resolution range for P1 box }
   generate                                 { generate reflections for P1 box}
   method=fft
   fft
      grid=0.25   { sampling grid for FFT and Patterson map (1/4 high resol.)}
   end
   update                                { compute Fcalcs for model in P1 box}
   do amplitude (fcalc=fcalc^2)        { compute |Fcalc|^2 and store in Fcalc}
   do phase (fcalc=0.0)
      map                  { compute Patterson map P1 (which will be rotated)}
                            { we write a hemisphere of Patterson vectors with}
      extend=box            { lengths less than $max_vector. }
      xmin=$m_max_vector xmax=$max_vector
      ymin=$m_max_vector ymax=$max_vector
      zmin=0.0 zmax=$max_vector
      automatic=true                           { use automatic scaling of map}
      formatted=false
      output=$p1_map                          { write an unformatted map file}
   end
end
xrefin                                     { make Patterson map P2 of crystal}
{===>}                                                { unit cell for crystal}
   a=67.4 b=67.4 c=290.1 alpha=90. beta=90. gamma=120.
{===>}
   symmetry=(x,y,z)       { operators for Patterson symmetry of crystal P622}
   symmetry=(-Y,X-Y,Z)
   symmetry=(Y-X,-X,Z)
   symmetry=(-X,-Y,Z)
   symmetry=(Y,Y-X,Z)
   symmetry=(X-Y,X,Z)
   symmetry=(Y,X,-Z)
   symmetry=(X-Y,-Y,-Z)
   symmetry=(-X,Y-X,-Z)
   symmetry=(-Y,-X,-Z)
   symmetry=(Y-X,Y,-Z)
   symmetry=(X,X-Y,-Z)
{===>}
   nreflections=20000
   reflection @hexpep.xpl end                { read reflections }
{===>}
   resolution 20 3                                    { resolution range }
   reduce
   do amplitude ( fobs = fobs * heavy(fobs - 2.0*sigma)) { sigma cutoff }
   fwind=0.1=100000
   method=fft
   fft
      grid=0.25          { sampling grid for Patterson maps (1/4 high resol.)}
   end
   do amplitude (fcalc=fobs^2)          { compute |Fobs|^2 and store in Fcalc}
   do phase (fcalc=0.0)
   map                                             { compute Patterson map P2}
      extend=unit
      automatic=true                           { use automatic scaling of map}
      formatted=false
      output=$p2_map
   end
end
xrefin
   nrefl=10                                             { release some memory}
   search rotation
      p1input=$p1_map    formatted=false
      p2input=$p2_map
{===>}
      range=5.0 $max_vector           { Patterson vector selection for map P1}
      threshold=0.0
      npeaks=15000                      { use 15000 largest vectors of map P1}
{===>}
      tmmin=0.0 tmmax=60.           { Lattman angle grid.  Specify asymmetric}
      t2min=0.0 t2max=90.           { unit for rotation function here.  See}
      tpmin=0.0 tpmax=720.          { Rao, S.N. et al. (1980). Acta Cryst.A36}
                                    { 878--884. }
      delta=2.5
{ Roughly, delta should be less than ArcSin[ high resol / (3*$max_vector)].}
      list=hexpep.rf                { output file for cluster analysis }
      nlist=6000            { analyse highest 6000 peaks of rotation function}
      epsilon=0.25                         { matrix norm for cluster analysis}
   end
end
stop
\EOD

4.5.4. Refine all the RF solutions by the PC method

#
xplor  <<\EOD                 # execute, using control data up to \EOD
remarks file xtalmr/filter.inp   -- pc-refinement of rotation function peaks
 parameter
   @topPAR:parhcsdx.pro                      { Read empirical potential }
                                             { parameter file CHARMM19 }
 end
{===>} structure @penpep.psf end             { read structure file }
{===>} coor @penpep_xplor.brk                { read coordinates }
evaluate ($wa=10000.)    { this is the weight for the XREF energy term }
                         { in this case it is arbitrary since we're not }
                         { combining it with other energy term }
xrefin
{===>}                                                { unit cell for crystal}
   a=67.4 b=67.4 c=290.1 alpha=90 beta=90 gamma=120
{===>}
   symmetry=(x,y,z)                  { operators for crystal symmetry P6(5)22}
   symmetry=(-Y,X-Y,2/3+Z)
   symmetry=(Y-X,-X,1/3+Z)
   symmetry=(-X,-Y,1/2+Z)
   symmetry=(Y,Y-X,1/6+Z)
   symmetry=(X-Y,X,5/6+Z)
   symmetry=(Y,X,2/3-Z)
   symmetry=(X-Y,-Y,-Z)
   symmetry=(-X,Y-X,1/3-Z)
   symmetry=(-Y,-X,1/6-Z)
   symmetry=(Y-X,Y,1/2-Z)
   symmetry=(X,X-Y,5/6-Z)
   SCATter ( chemical C* )
      2.31000 20.8439 1.02000 10.2075 1.58860 .568700 .865000 51.6512 .215600
   SCATter ( chemical N* )
      12.2126 .005700 3.13220 9.89330 2.01250 28.9975 1.16630 .582600 -11.529
   SCATter ( chemical O* )
      3.04850 13.2771 2.28680 5.70110 1.54630 .323900 .867000 32.9089 .250800
   SCATter ( chemical S* )
      6.90530 1.46790 5.20340 22.2151 1.43790 .253600 1.58630 56.1720 .866900
   SCATter ( chemical P* )
      6.43450 1.90670 4.17910 27.1570 1.78000 0.52600 1.49080 68.1645 1.11490
   SCATter ( chemical FE* )
      11.1764 4.61470 7.38630 0.30050 3.39480 11.6729 0.07240 38.5566 0.97070
{===>}
   nreflections=100000
   reflection @hexpep.xpl end                { read reflections }
{===>}
   resolution 20 3                                    { resolution range }
   reduce
   do amplitude ( fobs = fobs * heavy(fobs - 2.0*sigma)) { sigma cutoff }
   fwind=0.1=100000
{===>}
   method=fft
   fft
       memory=2000000                      { fft method with memory statement}
   end
   wa=$wa
   target=E2E2                                               { specify target}
   mbins=20                           { number of bins used for E calculation}
   tolerance=0. lookup=false                 { this makes the minimizer happy}
                           { expand data to a P1 hemisphere: this sequence of}
                           { statements first applies the crystal symm. ops}
                           { to the current reflections.  In the second step}
                           { Friedel mates or other redundancies are removed.}
                           { This is necessary since the application of the}
                           { symmetry operators can produce Friedel mates }
                           { under special conditions. }
   hermitian=false  expand  hermitian=true  symmetry reset  reduce
end
flags exclude * include xref end                  { only use XREF energy term}
{===>} set display=hexpep.fil end       { write the results of the refinement}
                                        { to a file called "filter.list" }
set precision="5" end
set message=off end                    { turn off messages and echo to reduce}
set echo=off end                       { output }
evaluate ($number=0)
evaluate ($counter=0)
                                    { loop over all orientations as specified}
                                    { in file rotation.rf (conventional rf)}
for $1 in ( @hexpep.rf ) loop main
   evaluate ($counter=$counter+1)                 { this series of statements}
   if     ($counter=1) then  evaluate($index=$1)  {assigns the information of}
   elseif ($counter=2) then  evaluate($t1=$1)     { a single line in file }
   elseif ($counter=3) then  evaluate($t2=$1)     { rotation.rf to approp.}
   elseif ($counter=4) then  evaluate($t3=$1)     { variables.  A single line}
   elseif ($counter=5) then                       { contains }
     evaluate ($rf=$1)                            { $index $t1 $2 $t3 $rf. }
     evaluate ($counter=0)
     evaluate ($number=$number+1)
     coor copy end                             { save current coordinates }
     coor rotate euler=( $t1 $t2 $t3 ) end     { and then rotate them }
                                               { according to the orientation}
                                               { specified by $t1, $t2, $t3}
     energy end                                      { compute initial energy}
     evaluate ($pc1=1.0-$xref/$wa)                   { and store in $pc1 }
     minimize rigid                         { rigid body minimization of the}
        nstep=15                            { orientation of the molecule }
        drop=10.
     end
     evaluate ($pc2=1.0-$xref/$wa)
                                            { rigid body minimization of two}
{===>}                                      { monomers. }
     minimize rigid
        group=( resid 3:190 or resid 299:321)
        group=( resid 191:298)
        drop=40.0
        nstep=40
     end
    evaluate ($pc3=1.0-$xref/$wa)
    coor swap end                { fit coordinates to starting structure in}
    vector do (vx=x) ( all )     { order to measure the orientation of the }
    vector do (vy=y) ( all )     { PC-refined structure }
    vector do (vz=z) ( all )     { the arrays vx, vy, vz are used as temp. }
    coor fit end                 { stores in order to keep the starting }
    vector do (x=vx) ( all )     { coordinates }
    vector do (y=vy) ( all )     { the COOR FIT statement stores the angles}
    vector do (z=vz) ( all )     { in the symbol $theta1, $theta2, $theta3 }
                                 { print information: orientation of rotation}
                                 { function peak ($t1, $t2, $t3), orientation}
                                 { after PC-refinement ($theta1, $theta2, }
                                 { $theta3), index of the rotation function,}
                                 { rotation function value, PCs for initial,}
                                 { rigid body and domain refined structures.}
   display $t1 $t2 $t3    $theta1 $theta2 $theta3    $index $rf $pc1 $pc2 $pc3
   end if
end loop main
stop
\EOD

4.5.5. Plot the results

The plot shows the PC values for each RF solution before and after the segmented-body refinement. The correct solution (# 34) has the highest PC value both before and after refinement, though the refinement actually reduces the discrimination of the correct solution relative to noise solutions.

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