4. Solvent-flattening, map calculation and display
Now that we have calculated a multiple isomorphous replacement (mir) phase set, we can Fourier Transform this to obtain our first electron density map. You will see that this map is quite noisy, and in fact is quite difficult to interpret. Download the coordinates for the refined structure (for reference and for centering the maps) here (the Nucleic Acids Database) or here (same file locally). We'll superimpose this on the map to help you see what is going on.
A. Calculation of Electron Density Maps
First, let's calculate an electron density map using the phases we just calculated with mlphare in Part 3. We'll use the CCP4 program fft to do this, and then also extend the asymmetric unit of the map using the program mapmask so that we are looking at a block of density that includes (but is not limited to) the molecule in the above pdb file (urx057.pdb).
Dowload this c-shell script, mir_map.csh, and read it. We want to be sure to load in the Fobs for the native structure and its associated sigmaF values. In addition, we want to load in the best estimates from mlphare for the phases, PHIB, and the associated confidence measures, or figures of merit, FOM, that weight each contribution to the fourier summation from 0.0 to 1.0 depending upon our degree of confidence in the phase estimate. We do this in the line that looks like this:
LABIN F1=Fnomnph5 SIG1=SIGFnomnph5 PHI=PHIB W=FOM
In calculating an electron density map, we also want to use all of the low-resolution data, so our resolution range will be
RESOLUTION 99. 3.1
in order to include all of the data. The log file will be pretty boring to look at, so just run the program by issuing
% chmod +x mir_map.csh
% mir_map.csh
and inspect the output to the screen to be sure the program ran. It will produce one map that says delete_me.map, which isn't of much use, and then the extended map that covers the molecule, urx057_mir.map . It is the latter that we want to look at.
It might be easiest at this point to use the program pymol to observe the map. Install it now if you haven't already got it. Now download the file mir_block.pml as well as the coordinates above if you haven't already. (Tutorials for using pymol on OSX and Windows are here; the linux version, once installed, more closely resembles the windows version.)
If pymol is configured properly, you can launch it and run the above script simultaneously just by typing
% pymol mir_block.pml
[Note that if you used MY install script for OS X native pymol, it needs to be invoked with npymol; if you used fink on OS X to install the X-windows version (which fink does with fink install pymol, the above works but the graphics display is not nearly as nice.)]
Otherwise, start pymol, and give the full directory address to mir_block.pml at the PyMOL> prompt on the screen, i.e.,
PyMOL> @/Users/mydirectory/tutorialdirectory/mir_block.pml
You'll get a screen with a lot of blue electron density on it. It will be quite noisy, but you might be able to see helices. As an aid to the eye, go to the "internal GUI" in the display window, find the line that says "initial" which is the name of the molecule, and go to the little square with an S on it, and put the mouse cursor arrow on it, hold down the mouse, and select "sticks," the third option from the top. This will display the molecule on top of the electron density. Move the display around to get a good look at the map, and then when you are done, you can get out of the program with
PyMOL> quit
B. Solvent Flattening
The above should convince you that the map could use some serious improvement. We can use the technique of solvent flattening (noise suppression) to improve the quality of the phases and therefore the electron density. Even with the above map, it is reasonably clear which parts of the density belong to the model and which parts are noise. By drawing a "mask" around the molecule, we can create a low-resolution model of our structure, and cancel the density peaks outside of it. As long as we get the low-resolution mask right, this will improve the quality of the phases and the electron density. We can use a variety of approaches to do this. One of the simplest is implemented in the CCP4 program dm. A more sophisticated approach is used by the program Solomon, which you can try using this script as a template if you wish. For now, try this simple dm shell script, dm.csh. After you run it, you will have a new phase set file urx057_dm_phases.mtz that contains everything that the previous phase set included, but now has two new sets of entries, PHI1, the new solvent-flattened (and improved) phases, and W1, their new figure of merit weights.
The new map calculation shell script, dm_map.csh, is essentially the same as before, except we tell the program to read in the following:
LABIN F1=Fnomnph5 SIG1=SIGFnomnph5 PHI=PHI1 W=W1
Run it as before, and then use pymol to display the electron density with the pymol script file dm_block.pml exactly as before. You will immediately notice that the density is of a much higher quality. Again, you can display the molecule on top of the density as described in Part A.
% pymol dm_block.pml
Finally, after you have finished this, you can view the map and molecule with just the map adjacent to the molecule displayed, using the pymol script dm.pml
% pymol dm.pml
It is an understatement to say that the density is much more interpretable.
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