Analysing an active
Building a functionnal unit
from a monomer
Electron Density Maps
Fitting Residues into
31 March 98
maps : lysosyme
To complete this tutorial,
you will need to download some material (coordinates and
electron density map of the lysozyme).
The data set has kindly
been made available by Dr. Doug Ohlendorf. Nate Winter did a
rigid body refinement of the coordinates deposited by K.P.
Wilson, B.A. Malcom and B.W.Matthews (pdb entry 1hel) to
Doug Ohlendorf's data set.
- material for Macintosh (.hqx 986 Kb)
- material for PC (.zip 640 Kb)
The downloaded material
contains 5 files: the electron desity map (1hel.dn6),
coordinates of two slightly different models of lysozyme
(1hel.pdb and 1hew.pdb), a readme, and a model of lysozyme
in which four residues have been replaced with ALA
In this exercise,
you will use the map to identify the correct amino acid in
each case replace ALA with it, and fit it into the map.
Stereo detail of a part of the lysozyme
contoured at 2.0 sigma. Real-time rendering directly from
Step by Step
- First of all, if you are using a Macintosh, make sure
that Swiss-PdbViewer has at least 6000Kb of free memory
to run (click on the icon, press Command I, and adjust
the memory allocated to the program). Then launch it.
- Open the file "1hel.pdb"
- Then, verify that the checkbox "center upon loading"
of the "General preference" is not checked. If it
was checked, uncheck it, close the protein and reload
- Center the view by hitting the "=" key of the
numerical keypad right mouse button on the PC), and
enable the "Slab" item of the Display Menu.
- Click on the first button at the top left of the main
window and set the "slab depth" to 8Å in order to
limit the quantity of information displayed on screen at
the same time.
- Select the Open DN6 Map from the file menu and load
the file "1hel.dn6".
A dialog will appear. The upper part provides some
information about the unit cell size: the length along
its axis, and the angle between the axis.
- Choose the "Display around CB" radio button, set the
value to 6, 6, and 6 and contour at 1.0 sigma.
- Load the protein 1hew.pdb and make it active
- Colour it by RMS. Almost everything will appear dark
blue, meaning that superposition is perfect. But one
region is coloured more brightly.
- Bring up the align window, select the zone coloured
by option clicking (right mouse button on PC) and hit the
return key to display only the selected residues.
- Control click (Shift Control-Click for the PC) on the
Pro70 residue of 1hel which will center its CA in the
view. Inspect the region. The backbone is flipped. This
region differs between the two pdb entries. Colour all by
B-factor, and observe that once more this is a region
with relatively high B-factor.
- Close the protein 1hew.pdb, and redisplay every amino
acid of the 1hel.pdb protein.
- open the protein "mutant.pdb" As you can see, it is
not superposed onto 1hel. In fact it should be but as you
have previously rotated the protein to inspect it, the
referential has changed. close the protein "mutant.pdb",
use the item "reset orientation" of the edit menu and
immediately reload the protein "mutant.pdb". Observe that
the protein is now perfectly superposed.
This illustrate the importance of the RESET
orientation, that should be used whenever you save a
modification into a protein (unless the "save in Original
Orientation item of the File Menu is enabled, in which
case it will be done automatically). Otherwise when you
reload it, it will no longer fit into the electron
- Hide the "1hel.pdb" layer by disabling the visible
box of the control panel, and
switch to the "mutant.pdb" layer.
- Center the view on Ala 38 (as you did previously with
Pro70). Note that a blob of electron density is not
occupied by any atoms. Try to figure out which residue
should be put at this location (without cheating by
looking at the solved structure!). Click on the mutate
tool in the main window, click on any atom of the Ala 38,
and browse the rotamer library of your suspected residue
(by hitting the * key of the numerical keyboard). Accept
the mutation of the rotamer that fits more closely to the
electron density. No rotamer fits perfectly, and you will
have to tweak the torsion angles. Click on the
torsion tool and hold down the
key number 2 while clicking and moving the mouse. It will
rotate the bound binding the CB and the CD atoms. Adjust
at best the sidechain within the electron density (you
might have to use also the "1" key to rotate around the
CA-CB bond. Once you are satisfied, accept the
- Repeat the process for residues 59, 64 and 66.
Then make the "1hel.pdb" layer visible and compare
your work with the solution.
Note: in real life, after having tweaked the
residues, The atoms positions are refined again with
X-PLOR, and a new
inspection of the map and tweaking is done until
everything seems to fit properly.