For next steps see the picture here

1.1 Change to the Coordinate Utilities module.  Select the Cell Content Analysis task.

1.2  Select the MTZ file - the program will read the space group and cell dimensions from the MTZ file (so you do not need to type them in):

MTZ file             DATA         cardiotoxin.mtz.

1.3 Enter the molecular weight of the protein.  The protein has 60 residues and we say average residue weight is 100 Dalton.  So

Molecular weight of protein 6000.

1.4 Click the Run Now button.

1.5 Look at the output in the window - it shows a table of the Matthew's coefficient and percentage solvent content dependent on the number of molecules that are in the asymmetric unit.
 

For estimated protein molecular weight 6000

Nmol/asym  Matthew's Coeff %solvent

  1         6.6             81.2

  2         3.3             62.5

  3         2.2             43.7

  4         1.6             24.9

  5         1.3              6.2

1.6) Close the Cell Content Analysis window.

Stage 2)  Look at the Experimental Data

a) Create a Patterson map and search it for peaks.  We expect a big peak at the origin (position 0,0,0) but if there is another big peak (perhaps about 0.25 the size of the origin peak) then perhaps there is  translation between the molecules in the asymmetric unit and it will be more difficult to solve.

(The theory behind this is explained on the web site of Bernhard Rupp: http://www-structure.llnl.gov/xray/101index.html For more information, go to the section on Phasing Techniques on this website, and click on NCS with native Patterson maps)

b) Create a Wilson plot which is an indication of the self consistency of the data.  Also find the average B-value of the data -  this can be used to help the molecular replacement program.

For the next steps look at picture here

2.1) Change to the Molecular Replacement module and select the Analyse Data for MR task.

2.2) Select the input experimental data

MTZ in      DATA      cardiotoxin.mtz

2.3)  Select input model:

PDB in   DATA     model.pdb

2.4)  Enter the Number of residues in the asymmetric unit - this is:

  number of molecules in asymmetric unit  *  number of residues per molecule

 =    3  *  60   = 180

Number of residues in asymmetric unit  180

2.5)   Run the job.  You can now Close the Analyse Data window.

2.6)  Look at the log file when the job has finished.  In the main CCP4i window click on the job called mr_analyse and then from menu View Files from Job select View Log File.  In the log file is output from the programs FFT which created the Patterson map and Peakmax which searched for  peaks in the map.  To find what we want click on the Find button and enter the text List of peaks.  You can now see table like this:

 Order No. Site Height/Rms    Grid      Fractional coordinates   Orthogonal coordinates

     1    1    1   38.66     0   0   0   0.0000  0.0000  0.0000     0.00   0.00   0.00

     2    2    2    3.34     4   0   0   0.0731  0.0000  0.0000     5.75   0.00   0.00

     3    7    3    3.10    25  16   5   0.4403  0.5000  0.1174    31.66  20.20   5.85

Hints In fact the values you get may be different, for example: Order No. Site Height/Rms Grid Fractional coordinates Orthogonal coordinates 1 1 1 58.43 0 0 0 0.0000 0.0000 0.0000 0.00 0.00 0.00 2 4 2 3.47 4 0 55 0.0546 0.0000 0.9821 -20.75 0.00 48.96 3 10 2 3.47 36 20 1 0.4454 0.5000 0.0253 34.40 20.20 1.26 but the comments above still apply. You may also see a different number of peaks, for example: Order No. Site Height/Rms Grid Fractional coordinates Orthogonal coordinates 1 6 1 38.66 28 16 0 0.5000 0.5000 0.0000 39.35 20.20 0.00 2 8 1 24.28 28 16 43 0.5000 0.5000 0.9773 14.42 20.20 48.72 3 5 2 3.34 24 16 0 0.4269 0.5000 0.0000 33.60 20.20 0.00 4 3 3 3.10 3 0 39 0.0597 0.0000 0.8826 -17.82 0.00 44.00 In this case x=0.5,y=0.5,z=0.0 is the centring operation of the spacegroup of the data (C2). It is a crystallographic translation of the origin peak (as opposed to a non-crystallographic translation). The difference is an effect of the width of the Patterson origin peak being related to the resolution range of data included when generating the Patterson map. At lower resolutions the origin peak may overlap neighbouring grid points in the map, and result in apparent extra peaks in these adjacent positions. Including higher resolution data narrows the origin peak and reduces the effect; try changing the high resolution limit from 4.0 A to 3.0 A in the Define Map folder, and re-run.

2.7)  Now go to the bottom of the log file where you will see:

Average B value for experimental data = 18.178

Average B value for model = 20.000

Running aMoRe: set the Tabling parameter BADD

(the amount to add to the Bvalue) to -1.822

Wilson Plot

This is a usual Wilson plot - no problems here!

Amplitude Analysis v. Resolution

This plot is usual shape for amplitude versus resolution plot with 'water' peak at about 4A.

Average B v. Residue

This shows the difference from the mean - in this PDB file all the B values are set to 20.   This is not interesting for this protein.

Quit from the two windows which display the log file and the graphs.
 

Stage 3) Run MolRep Molecular Replacement Program

For the next few steps look at picture here

3.1)  From the Molecular Replacement menu select MolRep - auto MR

3.2)  The default mode for running MolRep is good:

Do molecular replacement performing rotation and translation function

3.3) Select the input experimental data file.

MTZ in DATA    cardiotoxin.mtz.

3.4) Select the input model.

Model in   DATA     model.pdb

3.5) Look down the window a little: set the

Search for 3 monomers in the asymmetric unit.

3.6) From the Run menu select Run Now.

MolRep will take a long time to run - if it is too long you can see the output files: $RESULTS/molrep.log  and $RESULTS/mr_model_molrep1.pdb.
Look at the log file by selecting View Any File from the right side of the main window, then select

Go to directory RESULTS

then

File molrep.log

and the click on Display and Exit

The log file lists many possible solutions.  After the rotation function:
 

 Number of peaks :      50

             alpha    beta   gamma   theta    phi    chi        Rf    Rf/sigma

 Sol_RF  1    28.27   60.29  182.91  148.91 -167.32  153.12    0.3796E+09  5.31

 Sol_RF  2    40.54   72.23  275.07  117.37  152.74   83.17    0.3249E+09  4.54

 Sol_RF  3   162.83   58.08  180.35  104.76  -98.76   60.26    0.3060E+09  4.28

 Sol_RF  4   325.42   64.83  249.36  146.36  128.03  150.77    0.3013E+09  4.21

 Sol_RF  5    64.03   63.07  262.23  115.31  170.90   70.70    0.2954E+09  4.13

After a translation function:
 

             alpha   beta    gamma   Xfrac  Yfrac  Zfrac Dens/sig R-fac   Corr

 Sol_TF_1  1   28.27   60.29 -177.09  0.091  0.000  0.297    3.91  0.598  0.139

 Sol_TF_1  2   28.27   60.29 -177.09  0.611  0.000  0.342    3.35  0.596  0.137

 Sol_TF_1  3   28.27   60.29 -177.09  0.174  0.000  0.105    3.23  0.602  0.125

 Sol_TF_1  4   28.27   60.29 -177.09  0.870  0.000  0.349    2.82  0.606  0.107

 Sol_TF_1  5   28.27   60.29 -177.09  0.268  0.000  0.471    2.67  0.607  0.120

The program runs the translation function three times to find three different molecules.  For the second run of the translation function it will take the best solution from the first run and try to find another molecule which will fit well with the first solution.  For the third run of the translation function it will keep the best solution from the first run and the second run and try to find molecule number three.

It is not possible to say what is a good score - this will depend on many things but it is good if the best score (the correlation factor in the column labelled Corr) is much bigger than the second best score.  When you are looking for several molecules the best score for the first (and perhaps second) molecule will not be very good but you hope that the best score for all three molecules is much better than for other possible solutions.
 
There are three molecules in the output PDB file - these have chain names: A, a and b.    At the bottom of the log file is some information about how these three molecules fit together.
 

Mol_1 Mol_2 Direction_Cosine_of_Axis      Angle     Rotation  Translation

A     a     0.0       0.1       1.0       86.8      124.3     1.3       0.1       0.0

A     b     -0.1      -0.1      -1.0      91.8      104.8     0.5       1.1       -0.1

a     b     0.0       0.0       1.0       85.6      131.3     0.9       0.8       0.0

3.7)  The output file mr_model_molrep1.pdb contains three copies of the input model moved to the right positions in the asymmetric unit.   The three molecules will pack together something like this.
 

Alternative Stage 3) Run aMoRe Molecular Replacement Program

Stage 4) Refine the Structure

For the next few steps look at the picture here

4.1) Go to the Refinement module and chose  Run Refmac5

4.2) Enter the name of the data file:

MTZ in        DATA            cardiotoxin.mtz

Enter the name of the input files - this is the coordinate file output from MolRep:

PDB in         RESULTS     model_molrep1.pdb

If you have not run molrep, use the model_molrep1.pdb file found in DATA:

PDB in         DATA     model_molrep1.pdb
 

4.3)  Now you must tell the program that it must keep the  non-crystallographic symmetry by keeping the three chains similar.  Click on the line with the folder title

Non-crystallographic Symmetry

4.4) Click on the button

Add non-X restraint

4.5) Click three times on the button

Add chain

4.6) Now set up like this:

Restrain together chain A
and B
and C
 

4.7) Make sure you are not creating a data harvesting file. Select:

Do not create harvest file

from the Data Harvesting section.
 

4.8) Run the program by clicking Run and Run Now.
 

4.9) You can look at a log file by using View Any File and selecting

Go to  directory        RESULTS

File type     log CCP4 log    filename filter   *.log

Viewer  View Log Graphs

and then select file:

File        mr_refmac.log

and then click on Display and Exit

Go to the bottom of the list of Tables in File and select

Rfactor analysis, stats vs cycle

 
 
The graph shows the Rfactor (red) and the Free Rfactor (blue) for 6 cycles of refinement.  The Free Rfactor goes down from 52% to 46%   The  Rfactor is high - this is normal after molecular replacement because we do not have a good model yet  but it goes down so we probably have a good solution.