This protein is now in the PDB as 1tgx.pdb (solved by A.Bilwes,B.Rees,D.Moras J.Mol.Biol. 1994, V239, p-122).
2) Look at our experimental data - are there any problems?
3) Run molecular replacement program to find solutions.
4) Refine the phases using NCS (non-cystallographic symmetry) phased refinement.
model.pdb -
contains coordinates of the model we will use to solve cardiotoxin.
cardiotoxin.mtz -
contains the experimental data.
Files in the directory $RESULTS
matthews.log
- the log file from Cell Content Analysis
mr_analyse.log
- the log file from Analyse Data
molrep.log - the log file
from Molrep
model_molrep1.pdb - the
output coordinates from Molrep
mr_refmac.log - the log
file from Refmac5 refinement
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 6.2We are looking for the right number of molecules to give about 50% solvent. It looks as if our crystal will have three molecules in the asymmetric unit but two molecules is also possible.
1.6) Close the Cell Content Analysis window.
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.
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(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 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 5.85The biggest peak is size Height/Rms 38.66 and is at position x=0,y=0,z=0 - this is as we expect. The next biggest peak is 3.34 It is much smaller so there is no translation (good!).
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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 -1.8222.8 Look at the graphs in the log file. From the View Files from Job menu select View Log Graphs. There are three tables in the log file - look at then in turn:
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.
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 4.13This shows the possible rotation of the molecule: alpha beta gamma (or theta phi chi in polar coordinates). The score for the solution is the Rfactor.
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 0.120This shows the rotation (alpha beta gamma) and translation as fractional coordinates (Xfrac Yfrac Zfrac). There are different ways to score the solutions density/sigma, Rfactor, Correlation coefficient - the correlation coefficient is the best - this is bigger for good solutions.
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 0.0The diagram below shows what this means: there are three molecules and the vectors between the centres of the molecules. The direction of the axis of rotation to map one molecule onto another is shown; it is at 90 degrees to the vector between the molecule centres. To map one molecule onto the other needs some translation and a rotation of approximately 120 degrees (in fact the angles are 131.3 degrees, 104 degrees and 131 degrees which are surprisingly different from 120 degrees). This is right for a three-fold rotation axis and this shows there is a rotation axis and not a screw axis. This helps to confirm that the solution is correct because MolRep did not use this information to find the solution.
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.
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:
Coords in
RESULTS 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) Run the program by clicking Run
and Run Now.
4.8) 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.
Prepared by Liz Potterton (lizp@ysbl.york.ac.uk) &
Eleanor Dodson, July 2000
Additional material: Peter Briggs & Martyn Winn, February 2001