Molecular Replacement Tutorial

Alternative Stage 3) Run AMoRe Molecular Replacement Program

A3.1) From the Molecular Replacement menu select AMoRe.

A3.2) There are two windows - in the Amore Model Database window enter the file name of the model coordinate file.  For Trial model coords select the directory DATA and the file mr_model.pdb.   Notice that the model is given the name mr_model.  You can now Close this window.

A3.3) In the AMoRe window you will see that the default mode is Auto-Amore which will run through all of the AMoRe functions to find the solutions for one molecule.  The input trial model is also set to mr_model.

A3.4) In the AMoRe window enter the name of the experimental data file.

MTZ in   DATA    cardiotoxin.mtz

A3.5) Click on the grey folder title line Sort and Tabling Parameters to open the folder.  In the second line of the folder put

 add-1.8 to input Bfactors

A3.6) Run the program.

A3.7) When finished look at the output files with View Files from Job.  You will see that there are three output files called ...rot_mr_model.mr (the rotation solution),  ...tran_mr_model.mr (the translation solution) and ...fit_mr_model.mr (the fitting solution).

Hints The "..." in the output file names above will be something like TEST_3_ (so you will have TEST_3_fit_mr_model.mr, for example). The naming convention is for the first word to be the Project alias (in this case TEST), and the number to correspond to the number of the job in the main window (in this it would have been job number 3). In the steps below, each run of AMoRe will generate new rot, tran and fit files, and at each stage you will need to use solutions from the last (i.e. most recent) run of AMoRe. So to distinguish output from each run and get the correct file, check the number in the file name.

Look at the 'fit mr' file (that is the one called ...fit_mr_model.mr).  This file contains the best solutions for finding one molecule- the rotation angles are in columns 2 to 4 and the translations in columns 5 to 7.  The goodness of the solution is indicated by the correlation coefficient in column number 9.  You see there are several solutions with very similar correlation coefficient.
 

#CCP4I VERSION CCP4Interface 1.2 development

#CCP4I SCRIPT MR fit mr_model

#CCP4I DATE 04 Jul 2000  15:37:17

#CCP4I USER lizp

#CCP4I PROJECT devtest

#CCP4I JOB_ID 10

SOLUTIONF_1    1   57.88   76.14  145.84  0.3914  0.0000  0.2780 37.2 55.3 27.5   1

SOLUTIONF_2    1  295.14   69.11  149.56  0.0397  0.0000  0.1042 36.7 55.4 28.0   2

SOLUTIONF_3    1  162.25   67.79  149.59  0.0862  0.0000  0.2886 35.2 55.8 24.6   3

SOLUTIONF_4    1   34.78   66.32  149.23  0.0758  0.0000  0.1282 34.0 57.6 32.1   4

SOLUTIONF_5    1   52.63   77.37  343.33  0.3363  0.0000  0.4313 33.9 57.1 27.5   5

#SOLUTIONF_6    1  225.16   74.66  351.05  0.4542  0.0000  0.3425 34.2 56.8 28.9   6

#SOLUTIONF_7    1  296.25   82.29  320.95 -0.0033  0.0000  0.3849 31.8 56.6 28.6   7

Click the Quit button.

A3.8) Go back to the AMoRe window and change from running auto-AMoRe to running just a translation function to find the second molecule in the asymmetric unit.

run translation function

To run the translation function we need to know the rotation solutions - so

Rotation solution file TEST       ..rot_mr_model.mr

Click on the View button for this file.  You will see the file contains many solutions - to be quicker we will use just five.  If we put a # at the beginning of a line in this file then the solution will not be used.  Click on the button Change All - there will be # at the beginning of every line.  Now click on the first five lines - the # will be removed from these line.  Now click on Save and Exit

A3.9)  We want AMoRe to look for another solution assuming that we already have one solution.  We must tell AMoRe where to find the 'known' solutions.  Click on the button Add known solutions - this makes an extra line in the window, select:

Solution file   TEST     ..fit_mr_model.mr

This file contain many solutions and we want to  try just the best five.  Click on the View button for this file.  Click on the button Change All - there will be # at the beginning of every line.  Now click on the first five lines - the # will be removed from these line.  Now click on Save and Exit

A3.10) Run the job.

A3.11) The output from this job is then put into another  run of AMoRe.  Go back to the AMoRe window and change the name of the input solution file to the output file from the last job.

Solution file  TEST  ..tran_mr_model.mr

A3.12)  Look at this solution file - click on the Browse button.  The start of this file look like this:

SOLUTIONTF1_1   1   57.88   76.14  145.84  0.3914  0.0000  0.2780 37.2 55.3  0.0   2   22.9

SOLUTIONTF2_1   1  293.37   67.11  149.61  0.3251  0.4350  0.2701 30.0 54.9 15.5   2   27.8

SOLUTIONTF1_2   1   57.88   76.14  145.84  0.3914  0.0000  0.2780 37.2 55.3  0.0   2   22.9

SOLUTIONTF2_2   1  160.24   67.99  148.77  0.5868  0.3550  0.2890 26.5 56.5 16.3   2   32.2

SOLUTIONTF1_5   1   57.88   76.14  145.84  0.3914  0.0000  0.2780 37.2 55.3  0.0   5   22.9

SOLUTIONTF2_5   1  293.64   83.07  320.90  0.5051  0.9665  0.3895 20.2 58.5 11.1   5   12.8

A3.13) Run the job.

A3.14) When the job has finished there is an output file ..tran_mr_model.mr with groups  of three lines which are three positions for three molecules.  It is a good idea to refine these solutions using the AMoRe fitting function.  So at the top of the AMoRe window:

run refine fitting

and select the last tran mr file:

Translation solution file  TEST   ..tran_mr_model.mr

Again it is good idea to use only a few of the solutions in this file to save time.   Click on the View button for this file.  Click on the button Change All - there will be # at the beginning of every line.  Now click on the first 12  lines - the # will be removed from these line.  Now click on Save and Exit

A3.15)  Run the job.

Unlike the output from MolRep, which automatically checks the packing and generates sets of coordinates in the correct orientation, AMoRe only outputs sets of rotations and translations. A separate task is provided to apply the transformations and check the packing of the potential solutions.

A3.16)  Select the Build AMoRe Output Model task from the Molecular Replacement menu.

A3.17) Use the refined fitting solutions from the last AMoRe run as input:

Fitting solution file TEST ...fit_mr_model.mr

The window will then redraw. From the fitting file it knows that there are three solutions, one for each expected molecule in the asymmetric unit, and that the coordinates are generated in each case by applying the rotations and translations from AMoRe to the original search model.

It is also necessary to supply an MTZ file which will give the task the correct cell parameters. At the bottom of the window:

MTZ in DATA cardiotoxin.mtz

Finally, enter a name for the output file which will contain the transformed coordinates:

Output coords TEST cardio_model.pdb

A3.18)  Run the job. The output consists of a number of pbd files with the transformed coordinates - the different 'molecules' in the structure are identified by different chain names: A,B,C and so on.

The task also runs the DISTANG program to check for problems with packing - so you should examine the log file to see the output of this step.

The next step is to Refine the Structure.