(If you are resuming the tutorial after a break, start your desktop copy of RasMol, arrange the windows as in section 1, and open 3b5c.pdb. Restrict the view to residues 64-72, mainchain only. Turn on hydrogen bonds.)
Display: Spacefill
Rotate this model to view it end-on. Notice that stick models
make protein structures appear very open and empty, but even an
isolated helix is quite densely packed.
RasMol > restrict 64-72 < return
>
RasMol > hbonds off < return >
Display: Spacefill
Now you see a space filling model of the entire helix. The side
chains reappear because this restrict command includes
them.
RasMol > select < return >
Now all atoms are selected.
Display: Backbone
Colours: Structure
RasMol > reset < return >
This returns the full backbone to the screen, centers rotation on the
center of the whole model, and returns it to the original
orientation. The last four commands are useful when you get lost and
need to redisplay everything and get your bearings again.
RasMol > restrict sheet < return
>
This command removes all residues except those in pleated sheet
(sheet is another set). Center rotation on one of the
alpha carbons in the middle of the sheet (look back at previous
commands if you don't remember how, and remember to set picking back
to ident after you change the center of rotation). Display the
model as sticks and color it CPK. Display hydrogen bonds. To help you
see the sheet structure more clearly, remove the sidechains as
follows:
RasMol > restrict sheet and mainchain
< return >
Decide whether the three central strands in the sheet are parallel or
antiparallel. What about the edge strands? Are they parallel or
antiparallel to their neighbors? Here's how to check your
answers:
Display: Cartoons
Cartoon displays show sheet strands as arrows pointing toward the
C-terminal end of the chain. A pair of chains with arrows at the same
end are parallel. If arrows on neighboring strands are at opposite
ends, the strands are antiparallel.
RasMol > select < return >
RasMol > hbonds off < return >
Display: Cartoons
Colours: Structure
Now you see the whole protein as a cartoon. This is a vivid
display that is easy to interpret, but it has one disadvantage for
further exploration: picking doesn't identify atoms. Remember that
you must us a display function that shows the exact location of atoms
in order to identify atoms by picking.
RasMol > restrict turn < return
>
Display: Backbone
Now you see the beta turns in the model.
RasMol > restrict 17-21 < return
>
This shows just one turn. Display it as sticks in CPK colors, and
turn on hydrogen bonds. Notice that residue 19, the middle residue,
is lysine.
RasMol > center lys19.ca < return
>
Confirm that the hydrogen bond in a beta turn connects the carbonyl
oxygen of residue n with the N-H of residue n+3. From
information in a standard biochemistry textbook, decide whether this
is a type I or type II turn. One way to look at it: if the three
carbonyls that make the turn (residues 17, 18, and 19) all point in
the same general direction (up or down when you look at the turn
edgewise), then it the turn is type I. If the middle carbonyl points
in the opposite direction from the other two, it's type
II.
RasMol > reset < return >
RasMol > select < return >
RasMol > hbonds off < return >
Display: Cartoons
Colours: Structure
RasMol > select hetero and not hoh
Display: Spacefill
Colours: CPK
Now you see the heme prosthetic group in its cartoon-protein
binding site. You will now use RasMol to explore the binding of the
heme to the protein.
Rotate the molecule so that you see the heme edge-on, protruding from the right- hand side of the protein. Notice that the binding pocket is composed of four alpha helices above and below the heme, and a four-strand pleated sheet on its inside edge. The molecule looks somewhat like a pair of jaws holding a heme. The teeth are four alpha helices, and the throat is pleated sheet. However, this cartoon view does not display any of the chemical details of heme binding.
RasMol > restrict within (7.0, hem) <
return >
Display: Wireframe
Colours: CPK
RasMol > center hem.fe < return >
RasMol > select hem < return >
Display: Ball & Stick
Now you see the heme as a ball and stick model surrounded only by
atoms that lie within 7.0 angstroms of heme atoms. The restrict
within and select within commands are powerful tools for
directing your attention to specific interactions within
macromolecules.
Look for possible electrostatic interactions and hydrogen bonds between the heme (ball & stick) and the protein (wireframe). First, look at the yellow iron (III) ion in the center of the heme. The ion is part of an octahedral transition metal complex. Four of its six ligands are the light blue nitrogens of the heme porphyrin ring. What are the other two ligands? Use picking to identify them. When you have learned the residue numbers of the side chains that contain the ligands, display them in stick form as follows:
RasMol > select (#1 or #2) and sidechain
< return >
(substitute the residue numbers of the ligands for "#1" and
"#2").
Display: Sticks
Now you should see two histidine side chains providing nitrogens
as the fifth and sixth ligands of iron(III). The stick display does
not include the alpha carbon in the main chain, because the alpha
carbon is not included in the selection sidechain, which
selects CB but not CA. You can, however, include CA in the display,
and complete the stick model of the histidine side chains without
changing the selection, as follows:
RasMol > set bondmode or < return
>
Display: Sticks
Notice that one bond, the CA-CB bond, is added to the display for
each histidine. RasMol has two bond modes, called and
and or. In bond mode and, RasMol draws bond CA-CB if
both CA and CB are selected. In bond mode or, RasMol
draws bond CA-CB if either CA or CB is selected. When you
start RasMol, the bond mode is and until you change it.
Consider your last two sticks commands, with CB selected and
CA unselected. On the first try, in the and bond mode, RasMol
did not draw the the CA-CB bond as a stick because CB was selected
and CA was not. Then on the second try, in the or mode, RasMol
drew the CA-CB bond as a stick because CB was selected. Changing the
bond mode does not change the current display, but it does change the
behavior of subsequent display commands.
RasMol > set picking monitors <
return >
Working without stereo, click first on the iron (III) ion, and then
on one of its histidine nitrogen ligands. A dotted line appears
between the atoms, along with a label showing the distance between
the atoms in angstroms. The label is colored that same as the first
atom picked. If you accidentally pick the wrong atoms, you can remove
the dotted line and label by picking the same two atoms again. Now
measure the distance between iron (III) and the other histidine
ligand. This distance, about 2.0 anstroms, is the length of the bond
between iron (III) and nitrogen in the transition-metal complex. Such
bonds were once called coordinate-covalent bonds, because one of the
two bonded atoms, in this case the ligand nitrogen, donates both of
the electrons to form the bond.
The heme has two chains that extend from the edge that sticks out of the binding pocket. Both chains end with carboxyl groups. Can you find any heme-protein interactions that involve the heme carboxyls? There are two hydrogen bonds involving serine 64. Find and measure them. To make all your measurements easier to see, use the cursor-up key to retrieve and execute your command restrict within (7.0, hem). Then display all the atoms of the heme and its binding pocket in wireframe. Another quirk of RasMol is that the heme iron is not displayed in wireframe. Select only the iron (hem.fe) it and display it as a ball using Ball & Stick.
Now you will study the remaining interactions that hold the heme in place, which are primarily hydrophobic.
RasMol > set picking ident < return
>
RasMol > monitors off < return >
This sequence resets picking to the default and removes all
measurement lines and labels.
RasMol > select hydrophobic and
sidechain < return >
RasMol > color yellow < return >
This sequence selects only the sidechains of hydrophobic residues,
and colors them yellow. The hydrophobic set is another set of
atoms you can use in select commands. The color command
does not add atoms to, or remove atoms from, the display. To see the
names of other colors you can use in commands, look at help
for color and colors. (In the colors help
information, RGB stands for "red-green-blue".)
RasMol > select hem.c?? < return
>
RasMol > color cyan < return >
This sequence selects all carbons in the hem and colors them
cyan. The question marks stand for unspecified
characters. The selection hem.c?? means, "atoms of heme
designated c followed by up to two additional unspecified
characters." Now you will display the heme and its binding pocket as
a space filling model.
RasMol > restrict within (7.0, hem) and not
hoh < return >
Arrange the model so that you are looking into the opening of the hem
pocket, with the two heme carboxyls pointing at you. It is much
easier to do it stereo. If you can't tell front from back, try
displaying in ball and stick, which gives better depth
cues.
Display: Spacefill
Now you see the heme peeking out of its pocket. One carboxyl
points out into space, and the other is in contact with two atoms of
serine 64. The cyan carbons of the heme are hydrophobic, as are the
yellow carbons of the hydrophobic sidechains. In this view, you see
hydrophobic interactions, therefore, as contact between cyan and
yellow. But much of the contact area is buried by the space-filling
models. Let's look inside.
RasMol > slab 100 < return >
Now hold down the control key at the bottom left of the
keyboard (not the command key). While holding the key
down, move the mouse pointer up the screen. As you move the mouse, an
invisible plane slides back through the model, cutting away
everything that lies in front of it. Thus you can slice into the
model to any depth, removing all foreground as you go. Slide the
pointer up and down the screen to change the position of this cutting
plane. (You may find that the action is choppy, because the computer
is doing many calculations to produce each successive view.) As you
cut into the model, notice the contacts between heme carbons (cyan)
and atoms of hydrophobic side chains (yellow). By releasing the
control key, you can rotate the model to cut into it from
other directions.
RasMol > slab off < return >
Again orient the model so you are looking at the heme peeking out of
its pocket.
RasMol > select hem < return >
Display: Wireframe
RasMol > select hem.fe < return >
Display: Ball&Stick
Now you can clearly see the interior of the pocket, and observe
its strongly hydrophobic nature. You can also see the two histidine
side chains that protrude into the pocket to interact with the iron
(III).
RasMol > select hem < return >
RasMol > dots 200 < return >
This display colors the surface of the heme with about 200 dots per
atom. Dot displays give a feeling of solidity, but you can see
through them to neighboring atoms. Zoom in and try to see contacts
between heme carbons and other atoms in the pocket. These contacts
are much easier to see in stereo. For a dramatic view of the pocket,
try turning the model around and slabbing in through the back. You
will see what the world looks like from the perspective of the iron
(III) ion.
File: Close
Cytochrome b5 is a small protein consisting of a heme and only one polypeptide chain. In your biochemistry class, you will also study oligomeric proteins, which consist of more than one polypeptide subunit, as well as protein-protein and protein- nucleic acid complexes. RasMol has some features that are very useful with models that contain more than one chain. Now you will briefly examine such a model -- part of an antigen-antibody complex.