It is important that you familiarize yourself with MAGE by working through demo5_4a.kin before beginning this tutorial.
The tutorial example described on the following pages lets you practice by constructing a kinemage to illustrate the structural features of ricin (Protein Databank file 2AAI.pdb). This tutorial leads you through many of the authoring functions for making your own kinemages, including stylistic suggestions on how to choose views, colors, button layouts, etc. in ways that will help communicate your 3-D ideas effectively. Several more specialized functions, such as rotatable bonds, are included in the later parts. If you want to work on kinemages at home you can find all the information and programs you need at http://orca.st.usm.edu/~rbateman/kinemage/authorlinks.htm .
This tutorial is designed for MAGE and PREKIN version 6.30 and higher. Students are encouraged to quickly view the PREKIN tour before beginning the tutorial.
KINEMAGE-CONSTRUCTION TUTORIAL - RICIN
: Ricin is an extremely potent toxin capable of crossing membranes and inactivating ribosomes by cleavage of ribosomal RNA. Because of its potency, it has historically been used in espionage, chemotherapy, etc. It is composed of two unrelated polypeptide chains, with the catalytic site in the A chain (killer subunit), the cell recognition site on the B chain, and four bound carbohydrates. The crystal structure of the whole molecule was determined by Jon Robertus and colleagues at the University of Texas, and as stated above, is PDB file 2AAI.pdb
KINEMAGE 1- Overview of the molecule
To start - a simple alpha carbon backbone kinemage
For an initial look at the ricin molecule, we will run a simple default script on file 2AAI.pdb. Launch (i.e. start) PREKIN and open 2AAI.pdb from its menu. PREKIN will ask for an output file name, so call the output file backbone.kin. When the first dialog box of choices comes up, accept the default "Backbone browsing script", which will execute a simple script producing Calphas (a connected series of alpha carbons), disulfides, and non-water het groups for all subunits in the file. A ‘het’ group is anything not part of the polypeptide (or polynucleotide) chain. When PREKIN is done, it will launch MAGE so you can look at the resulting kinemage.
You should see Calpha backbones for the two ricin chains in different colors, with yellow disulfides and several bound sugars (pink). Move the image around by dragging with the mouse. Such a simple, default kinemage shows many of the features of the structure, and is useful for many purposes. Note, however, that the viewpoint is arbitrary and the default colors, names, and arrangement of buttons are not ideal. If you want to show particular details and express your point clearly to someone unfamiliar with the structure, then there are many ways to make the kinemage more informative and persuasive. You are the artist and the kinemage is your canvas, so take the time to examine each image you create from the point of view of both artist and scientist.
Choosing and saving views in MAGE
In this section you will prepare a 3-D portrait of the entire ricin molecule and similar portraits of each of the two individual subunits (chains).
Creating an overview: First move the image around to find a view that spreads out the three domains in the plane of the screen, with the A chain (the white one) at the top. See if you can enlarge the zoom factor by one or two arrow-clicks on the zoom slider without going off the screen edges. Type ‘s’ on the keyboard to toggle into stereo, to make sure your zoom and orientation allow seeing most of the important parts in stereo (you can check for that, even if you can't see stereo yourself). Re-center if needed by checking the "pickcenter" box in the bottom right corner, clicking on the atom you want in the center of the screen, then unchecking the "pickcenter" box. Try using the keyboard keys ‘x’ and ‘z’ to move the center toward or away from you. Once satisfied, choose "Keep Current View" under the Edit menu, and save as View 1, giving it a descriptive viewID such as "overview". Move the image, then choose View1 under the View menu, which should reproduce the view you just saved.
Creating views of subunit features: Pickcenter near the middle of the A chain and zoom in somewhat. Choose the "Find" function under the Tools menu. In the dialog box, turn on pickcenter and ask to search for " 177 ", which is the active-site Glu of ricin; MAGE will center on Glu 177 and mark it. Turn off pickcenter. Choose a view for the A chain that shows both the central beta sheet and this active-site Glu, and save it as View2, with a viewID of "A chain". Now pickcenter between the two domains of the B chain, zoom in, and save a view that shows the domains fairly equivalently, in a vertical orientation to allow for stereo. It might help to unclick the A chain button so you can see the B chain better, although your view shouldn’t absolutely require that since your readers may not think of doing it.
Renaming and coloring groups: Turn on "Show Object Properties" (Edit menu), and click on any sugar. The resulting dialog box will show you the program's internal data structure for this item. Edit the group name to "sugars", and click on the "dominant" option below it (which will hide its subgroup and list buttons on the button panel). Accept the result, and see how the buttons have changed. In the same way, edit the group names for the protein, from 2aai 1 and 2 to "Ricin A ch" and "B chain" (remember that you only get about 11 characters for a group button name). Turn off "Show Object Properties".
Turn on "Change Color" (Edit menu), then click on a sugar atom. Pull down the color-choice list and release on white, and Accept. Now you need to change the colors of the two subunits, A to differ from the white sugars and B to contrast better with the yellow SS bonds. The "tint" colors work best for Calpha backbones, because they can be distinguished without overwhelming small features you want to emphasize. Make the A chain yellowtint and the B chain greentint (try out some other possibilities, too).
Saving your modified kinemage: Choose "Save as", under the File menu; you will be given a dialog box to place and name the saved file. Stick with the same name (backbone.kip). You will notice that the file ends in ".kip" rather than ".kin". The kip simply means that it is a modification of the original kinemage. Quit out of MAGE.
Adding the active site Glutamate: Use PREKIN to open the 2AAI.pdb file again, but this time name the output file "sidechain.kin". In the initial dialog box of PREKIN, choose "New Ranges". In the range dialog specify both start and end residue as 177, check the 'sc' (sidechain) and 'at' (balls for non-C atoms) boxes, the "OK accepts and ends ranges" button, and the OK button. In the following dialogs accept the defaults for no focus and 0.2A balls. In the last dialog window ask for "only first subunit" by making sure both entry windows have a 1 in them and the middle radio button is checked. When the program quits running, you have generated a side chain for Glu 177 of the A chain. DON’T EXIT YET! You now need to add the four sugar-linked Asn residues (46, 95, 135, & 255) of the B chain (subunit 2). Go to "New Pass" (File menu) and select "New Ranges". In the range dialog specify both start and end residue as 46, check the 'sc' (sidechain) and 'at' (balls for non-C atoms) boxes, then the OK button. The page will flicker, but return. Now change the start and end residues to 95, then click the OK button. Repeat for residue 135. For the last residue (255) you will need to additionally check the "OK accepts and ends ranges" button. Accept the defaults, EXCEPT in the last dialog box select subunits in the range 2 to 2 AND check the top radio button. When done, launch MAGE with this kinemage. You should see one Glu and four Asn sidechains floating in space. Exit MAGE.
Merging files in MAGE: Now launch MAGE with your backbone.kip file, then choose "Append" on the File menu to add in the sidechain.kin. This merges the two kinemages into one. Change the color of the Glu 177 sidechain vectors to something bright, contrasting, and oxygenish, such as pink or hotpink. Rename the appropriate 2AAI buttons as Glu 177 and Asns, respectively.
Using Drawline to draw sugar-Asn bonds:
Covalent bonds: Choose the B chain view and turn off (uncheck their buttons) everything but the Asn sidechains and the sugars. For each of the two long carbohydrate chains, find an Asn whose Nd2 atom (the blue ball representing the side chain nitrogen) is close enough to be covalently bonded (around 1.4Å) to a sugar atom. Turn on "Draw New" on the Edit menu and use "drawline" to add a line for each bond, by picking the two atoms.
Hydrogen bonds: Now select "Draw New Setup" (Edit menu), and in the dialog box set 0.7 in the "shorten lines" box, and check "line ends unpickable". Then, for each of the two-sugar chains, find an Asn whose Nd2 is H-bonded to an oxygen of a sugar atom (between 2.5-3.0 Å apart), and click on the two atoms to add a shortened line to represent the hydrogen bond. (Turn off "drawline" while measuring the distance; if you draw an unwanted bond remove it with "eraselast".)
Picking unpickable groups and hiding group buttons
When finished, turn off both pickcenter and drawline buttons. You will notice a "new group" button on the right, which refers to your newly-drawn bonds. Use "Show Object Properties" to change the name of "new group" to "bonds". If MAGE will not let you pick the group by clicking on a bond, turn on "superpick" (Edit menu) and click the end of a bond. If you want no button at all for the bonds, just check the "no button" box under the group name in the "show object properties" dialog box.
Look at the resulting kinemage to see if you did what you intended and whether you like the results. Modify views if needed, and note anything that needs to be changed during future editing. Save the modified kinemage as ricin1.kin.
KINEMAGE 2- Secondary Structure in the A Chain
Running the built-in ribbon script: Make
a new kinemage by launching PREKIN and again selecting the 2AAI.pdb. Save output in the
desired directory with the name “ricin2.kin”.
Select the “built-in scripts” menu, then the “ribbon: HELIX_SHEET”
option. Accept all defaults in the “ribbon” box. In the “run conditions”
box only select the first subunit (A chain) by
clicking the middle radio button and then OK. When done and before exiting, select “new
pass” under the file menu and again select “built-in scripts”. This time
select the “mcHb” script. OK and again select the first subunit in the “run
conditions” box. Launch MAGE at the end to see the created kinemage. You will have
two representations of the A chain overlayed. In the ribbon structure, beta
sheets should be green arrows and alpha helices gold spirals. Irregular secondary
structure, i.e. coils and turns, will be connecting ropes. In the mainchain structure you
will see the peptide backbone and hydrogen bonding.
Using
animations as alternate views of structure
Animating
between two different representations of an object can be very informative, even more so
if they have different conformations. You
have made two different representations of the ricin A chain: a line tracing of the
peptide backbone, and a ribbon diagram of the secondary structures. Next you will overlay and animate between these
two representations. MAGE allows animations
between groups simply by placing an asterisk in front of the group name.
KINEMAGE 3 - Active site, with hydrogen bonds and a rotatable sidechain
The philosophy behind kinemages is that the most revealing way to illustrate what is important is to remove extraneous parts of the structure. In this kinemage, you will concentrate on the active site of ricin, which resides solely in the A chain. To see what is going on at the active site, you will eliminate everything that is not within 12 angstroms of the critical active site residue Glu 177, which you highlighted in the first kinemage. Before beginning, I suggest you review your amino acid sidechains so that you will recognize them easily.
Generating an active site with the Focus option
The "Focus" option: Run PREKIN on file 2AAI.pdb again, with output file actsite.kin. Choose "Focus only". In the next dialog box, choose to do the focus on a residue by number. In the "Focus Point Values" dialog, specify residue 177; for radii try 8A for sidechains, 12A for main chain, and 0 for everything else; ignore the special logic controls. Do only first subunit.
Alternate method with atom balls: Run PREKIN on file 2AAI.pdb again, with output file actsite.kin. Choose “New Ranges” , then check the ‘mc’, ‘sc’, ‘at’ and the ‘OK accepts and ends ranges’ buttons. Hit “OK”. In the next dialog box, choose to do the focus on a residue by number. In the "Focus Point Values" dialog, specify residue 177; for radii try 8A for sidechains, 12A for main chain, and 0 for everything else; ignore the special logic controls. Do only first subunit.Setting up a rotatable residue: When PREKIN is done, choose "New pass", and ask for "New ranges". Set the residue range as 208 to 208, replace the 3 dots with "glu", and check the 'ro' box (rotation or mutation); this tells PREKIN to set up rotatable bonds for a sidechain. Click OK and end; no focus; first subunit. When it finishes, launch the kinemage.
Cleaning up the kinemage
Drawing hydrogen bonds: Choose and keep a view that gives a good close-up of the active site. Click on and identify the sidechains Glu 177 and 208, as well as the Gln, the two Tyr, the Arg, and the Trp that surround the two glutamates. In "Draw New Setup" set "shorten lines" to 0.7 and check "line ends unpickable". Turn on "Draw New", and draw in the Hbonds from the sidechains of Arg, Trp, and one Tyr to the closest mainchain carbonyl oxygens (the carbonyl of the peptide bond). It is helpful to zoom in on each side chain and measure the distances to the nearest oxygen. Remember that Hbonds are 3Å or less between atoms that share the hydrogen.
Pruning main chain: Now, using "prune" (Edit menu) trim away all extraneous main chain that is either floating in space in bits or nowhere near the sidechains or Hbonds. If you want, prune away any uninteresting sidechains also. (Save your file, then try out the "punch" , "prune", and "auger" buttons to see how each one works, using "undo p" to recover from mistakes. If you auger a hole through the best part, just reload the saved file.) Choose "Change Color" and pick an atom in the Glu 177 sidechain - but this time check the "point" color box before setting the color to, say, hotpink. Do this for each atom in the sidechain.
A Rescue Role for Glu 208?
Glu 177 is known to be the catalytic residue for ricin's nuclease activity on ribosomal RNA, and mutating it to Asp lowers that activity. However, much to everyone's consternation, the "control" mutation to Ala had nearly normal activity. Use the sliders at far right to rotate the sidechain dihedral angles of Glu 208 (there will still be a "ghost" sidechain at its original position), and see if you can get its carboxyl group close to the Glu 177 carboxyl position; could this happen in the Glu177Asp mutant? in the Glu177Ala mutant? An x-ray structure of the E177A mutant showed that this hypothesis is correct.
Save a view of each sidechain hydrogen bond, remembering to use descriptive titles for each view. Also save a closeup view of the overlap between the two glutamates. Finally, save the modified kinemage to your hard drive as ricin2.kin.
KINEMAGE 4- Superimposing the two domains of ricin chain B
Chain B has two domains that each have the "beta trefoil" fold; superimposing them can show how similar they really are. We will use the beta strands, the disulfides, and the Trp sidechains as landmarks for doing the superposition in MAGE with its docking function. (Also see alternative method with Deep View at the end of the document.)
You should see all of chain B, with Trp sidechains in cyan and SS in yellow. Turn off the second button on the button panel (domain 2) and use "Change color" to make the domain 1 Calphas white. Drag with the mouse down and then a little left to get a view down the 3-fold axis of domain 1 - you should see three Trp sidechains as symmetrical "T" shapes, with a triangle of Calpha strands evenly around a central opening. Type "x" 7 or 8 times to bring the molecule forward. Save this view as View1. There are two other Trp in domain 1 that are not symmetrically related around the 3-fold; use "Prune" to delete them. Turn on domain 2 and make its Calphas some other color (maybe pinktint). Use "Show object properties" to give the two groups names like "B dom1" and "B dom2". Find the 3 symmetrical Trp in domain 2, and prune away the extra, fourth one.
Domain 2 should be below and to the side of domain 1. The idea is first to rotate domain 2 into the same orientation as domain 1 and then to translate it until they overlap. Holding down the shift key, drag with the mouse to rotate domain 2 until you are looking down its 3-fold (with the 3 Trp symmetrical T shapes, and the Calpha triangle in back); then with the shift key down drag the mouse along the top of the screen to turn domain 2 in the plane of the screen until the orientation of its disulfides matches that of domain 1. [If you accidentally rotate without the shift key down, return to the reference orientation of domain 1 by choosing View1.] Once you are satisfied that the two domains show the same orientation, use the translation sliders in X and Y to move domain 2 on top of domain 1 (concentrate on the 3 Trp and parts of the beta sheet, since some of the loops are quite different). On the Tools menu, under "Empowerments", choose "Rotate y 90+", then use the X translation slider to line up the two domains in the third independent direction. Now drag without the shift key down, to evaluate the goodness of overlap. Make another round of adjustments if necessary, but don't try too hard to get it perfect.
Writing
text and caption
To add text to the text or caption
windows of any of your kinemages, do the following in MAGE. Under the Edit menu turn on
“text editable”. In the small text window on the bottom (caption window) replace
the default caption with a short 2 or 3-line description of what is shown and what the
colors mean. For example, in the large text
window in the top left of ricin1.kin put a title at the top in caps, your name as author,
then a blank line, " *{Kin 1}* - Ricin A and B chain, with Glu 177, SS, ribbons, and
sugars", another blank line, and a paragraph or so about what a reader should look
for in each view of the kinemage. (Omit
carriage returns except to end paragraphs, to let the text flow properly when windows are
resized.) The *{Kin 1}* in your Table-of-Contents entry is a hypertext link that will go
to kinemage 1 with (the default) view 1. To try it out, turn off “text editable”,
move to some other viewpoint, and click on *{Kin 1}*. Of course, this is more useful when
you have multiple kinemages to view. To place all four of your kinemages in one file,
refer to the section below entitled “Placing two or more kinemages in one file”.
What went wrong?
I double clicked the PDB file and nothing (or something strange) happened. The PDB file is just a text file and doesn’t do anything by itself. You have to either drag and drop it on PREKIN, or open PREKIN and select the PDB file.
I can’t find my file? I know I saved it! You probably saved it to a different directory accidentally. Use the "find" function in either Windows (under Start) or Mac (under File) to locate your file.
I have ribbons on both chains instead of just the A chain. You forgot to check the middle radio button on the subunit dialog box in PREKIN. (The default is to do all subunits.) The easiest thing to do is just to trash the ribbon kinemage you have and take a minute to make a new ribbon kinemage with PREKIN.
I get a blank graphics screen in MAGE when I open my kinemage. One possibility is that you are trying to open a PDB file (.pdb extension) instead of a kinemage (.kin or .kip extension). MAGE will not read PDB files. Another possibility is that you tried to make a kinemage with PREKIN using another kinemage as an input file. PREKIN will only use PDB files as input files. If your text box says something about EOF being reached, this second possibility is your problem. Start over and select a PDB file for PREKIN.
I put in the astericks but my animation still doesn’t work. The usual problem here is that there is a space in front of the asterick in the "show object properties" dialog box. Delete that space and your animation will work. Note that the animation is a simple switching between two structures, not a television production.
I made my sidechain.kin file, but it doesn’t seem to have any asparagines in it. It seems to have prolines and other stuff. Sounds like you either didn’t put 2 in both subunit boxes or didn’t make check the top radio button on the subunit dialog box in PREKIN when you made the Asn sidechains. It was probably still set for the first subunit based on the Glu 177 you did on the first pass. Trash the sidechain.kin and make it again using the correct subunit dialog box settings.
I want to change the "new group" name on my bonds, but I have turned on "show object properties" and can’t seem to select the bond. Try turning off the sugars and Asn sidechains, then clicking on the bond ends. If one bond doesn’t work, try another one. If none of them work, turn on "superpick" (Edit menu), then click on the bond ends.
My
markers disappeared. Markers are only active when you are using them.
They are not saved permanently.
When
I draw bonds, I am making lots of new groups. Why? Each time you turn on, then turn
off “Draw New” under the Edit menu, you create a new group. The best approach is
to decide what bonds you want to draw, turn on Draw New, make all of your bonds, then turn
off Draw New. This places all of the new bonds within a single new group.
My
PDB file contains a large number of structures. What do I do?
You probably have a structure that was solved by NMR, which often creates a large ensemble
of possible structures which fit the experimental data. If possible, try to find a single
“average” structure. If that is not available, just pick one of the ensemble to
work with.
When I create
views, save
them, and close the file, they aren’t correct when I reopen the file. This is a mysterious problem
and occasionally occurs when merging several kinemages into one file. It also
occurs when
using flatland to adjust an image prior to saving a view. If you are dragging
an image about using flatland, use pickcenter to fix the image prior to saving
the view. It is
always a good idea to recheck your views after making any significant changes.
If the views are not the way they should be and you want help, try to document
and reproduce what
happened and email me at robert dot bateman at usm dot edu.
I will try to reproduce the problem on my end and figure out what is going on.
Quick List of Frequently Used Kinemage Features
Remember to turn off a menu item whenever you are finished with it.
Measuring distances between atoms – In MAGE click on the first atom. At the bottom of the graphics window will be the identity of that atom. Now click on the second atom. The bottom of the graphics window will now contain both the identity of the second atom and the distance from the first to the second atom in angstroms.
Measuring dihedral angles – Turn on "Measures" (Mage Tools menu), then sequentially click the four atoms in the dihedral angle of interest. The dihedral angle will be displayed as the rightmost number at the bottom of the graphics screen. This number will always represent the dihedral between the last four atoms you click.
Coloring groups – Turn on "Change Color" (Mage Edit menu). Click desired group in graphics screen, choose color from menu, and accept. Turn off "change color".
Coloring connected groups - Make the domains separately with PREKIN using the New Ranges mode and multiple passes. They will then be separate groups that you can color separately.
Centering features – Turn on "pickcenter", click an atom of the feature you want centered, then turn off "pickcenter". Alternatively, you can hit the ‘f ’ key to enter flatland, then use your mouse to drag the image wherever you want. Hit ‘f ’ again to leave flatland.
Animating groups – Turn on "Show Object Properties" (Mage Edit menu), select the first group in the animation, and put an asterick in front of the group name. Do this for each group in the animation sequence. (Note: The asterick must be all the way to the left in the group box.)
Eliminating unneeded buttons – Turn on "Show Object Properties" (Mage Edit menu). Click the feature whose button you want to hide. This dialog box gives the hierarchy of group, subgroup, list, and point ID, with subgroups indented below their parent group and lists indented below their subgroup. Selecting "dominant" hides the buttons of all lower members. Selecting "no button" hides just that one button. Selecting "delete" will delete the entire group permanently.
Eliminating unwanted features – Turn on "Prune" (Mage Edit menu), then click on the atoms of the feature you want to remove.
Selecting (picking) a feature that won’t let me select it - Some features, such as new bonds, may be "unpickable". To pick them, turn on "Superpick" (Mage Edit menu). Then click the end of the bond. Turn off Superpick when finished. In general, remember you can only pick endpoints, not in the middle of lines.
Exploring a structure – To poke about a structure looking for interesting features, it is recommended that you make a kinemage using the ‘lots’ script in “New Ranges”. This will use all of the information from the PDB file to make one kinemage. It will be crowded, but you can turn groups on and off to explore.
Features of Interest to More Advanced Kinemage Authors
Kinemage authors who have completed the ricin tutorial and demo5_4a.kin should now complete demo5_4b.kin.
Saving the graphic image for a written report or PowerPoint presentation - Currently the best way is to do a screen capture and edit the image after pasting it into an image editor like Adobe Photoshop or Microsoft Image Composer. Mac users can also type "shift apple 4" to save a PICT file called "Picture 1" to the hard drive. For written reports, you should change the kinemage to a white background so you don’t use up an entire ink cartridge. Also, before capturing it is an advantage to zoom the kinemage to fill its window with the part of the image you want to see and tug at the corners of the MAGE image window to fill up the computer screen. MAGE can save images as postscript (.eps) files which can be read by Adobe Illustrator or printed with the Downloader utility (Mac). Sometimes they will print directly if you just drag the eps file to the printer icon. MAGE can also write files for input to the freeware rendering programs Raster3D or POV.Ray, but then you would need to get those programs and learn to use them. They can make the ribbons look especially nice.
Using kinemages interactively in a presentation - One easy way is to give your presentation in html using a web browser. If you have configured your browser to use MAGE as an application, you can set links in your presentation to kinemages on your computer. Setting links in PowerPoint slides works the same way. Alternatively you can simply use MAGE directly.
Configuring a browser to use MAGE - In Netscape, under Edit go to Preferences, then Applications. The file type is kinemage, the file extension is .kin, the MIME type is chemical/x-kinemage, and use the Browse button to point the application to your MAGE program. Internet Explorer has a similar setup protocol.
Putting labels on particular features - Labels in kinemages are copied from the
Point ID (what is shown at the bottom of the screen when you click on an atom or point).
Turn on "Draw New" , check the Labels box on the right of the graphic screen,
and pick a point to label it. If you click and drag, you can move the new label out away
from the point. To edit the label, turn off the structure (unless the label was moved),
turn on Show Object Properties, pick the label’s lower left corner, and edit what it
says in the rightmost field of the dialog box. It can be tricky to pick the label, but
keep trying. If you have trouble editing the label with MAGE, you can edit the point ID
before you make a label of it or you can always edit the text file directly (see below).
Changing the colors of the secondary structure ribbons - To change the color of all ribbons, turn on "Change Color" and click on one of the ribbons. The color you select will work for all of the ribbons.
Superimposing features – Decide what two groups you want to superimpose and
what you will use as reference points for the superimposition. Then turn one group off.
Turn on "Docking Scope" (Mage Tools menu), then turn the off group back on. The
sliders on the right will allow you to move one group relative to the other one. When
done, turn off "Docking Scope" and save the kinemage under a new name. This
is illustrated in kinemage 4 of the ricin tutorial. An alternative superposition method is
described at the end of this document (Superimposing PDB files using DeepView).
Making a specific mutation - This procedure is almost the same as simply setting up a rotatable residue. Open PREKIN with your PDB file (call the new kinemage "mutant.kin"), go to "New Ranges", set the residue ranges boxes to your particular residue number, replace the 3 dots with the three letter code of the mutant amino acid, and check the ‘sc" and 'ro' boxes (rotation or mutation). Click OK and end; no focus; and in the subunit dialog box clearly indicate the subunit on which the mutation will reside. When PREKIN finishes, You can open MAGE with your complete structure and append mutant.kin.
Adding atom balls to a ligand (cofactor, inhibitor, etc) - The strategy is to make
a separate kinemage of the ligand alone, then append it to the main kinemage. Open the PDB
file with PREKIN, go to New Ranges, and click only the "ht" and "at"
boxes. Then click the "OK accepts and ends ranges" button, hit OK and accept all
defaults. Launch the kinemage and you should have a ligand with atom balls. You can now
append this to the main structure in which the ligand has either been pruned away or the
structure made without het groups. If
you would like to make a space-filling rendering of a ligand instead of simply using atom
balls, click only the ‘ht’ and ‘ht CPKs instead of vectors’ in New
Ranges.
Creating
a focus around a ligand
– Repeat the preceding procedure, selecting the ‘mc’, ‘sc’,
‘ht’ and ‘at’ boxes. When the “focus options” dialog box
appears select ‘type in x,y.z for a focus point’, then “OK”. The next
dialog box will ask for the coordinates* and the diameter of the focus. Enter these, hit OK and accept all defaults.
*You
will have to obtain the XYZ points from a previously made kinemage by opening it with
MAGE, then Tools menu, XYZ point, and finally clicking on the center of the ligand.
Combining
hypertext and master buttons – Within
the text window a link can be set up to not only jump to a graphics image but also to go
to a particular view of that image and to turn on or off the master buttons associated
with that image. A good example of this is kinemage 3 of c1Basics.kin in the Branden and
Tooze (2nd ed) series. One of the hyperlinks here is * {kin 3 v=4 alloff
m={gly,pro} on, m={ca + mc} on} *
When
you click on this hyperlink, the image in kinemage 3 is opened to view 4 and only the
features in master buttons {gly,pro} and {ca + mc} are turned on.
Opening with certain
buttons on – If you save a kinemage out
of MAGE with particular buttons checked, those same buttons will be checked when you open
the kinemage again with MAGE. In other words, you can select the buttons for the opening
view simply by saving it exactly the way you want it to open. Buttons for other views are
only accessible via the hypertext/master button route.
Inserting mainchain into an alpha carbon backbone – The peptide bond often plays a role in binding interactions so it can be helpful to insert mainchain into binding sites when the rest of the strucure is kept simple with the Calpha backbone. This can be done by making multiple passes through New Ranges with PREKIN, selecting either ‘mc’ or ‘ca’ and inserting the start and stop residues for each pass. Alternatively, the complete Calpha backbone can be made, the desired sections of mainchain made in separate passes, and the overlapping Calpha backbone pruned away.
Editing the Kinemage Text File in a Word Processor
Some kinemage features require you to open your kinemage file (which is a plain text file) in a word processor or text editor and manually edit the text. This is a very powerful tool, but requires some familiarity with the kinemage file format (see demo5_4b.kin and KinFmtAuthors.txt). It is also very important that you save the edited file as plain text.
Placing two or more kinemages in one file
You probably noticed in many premade kinemages that there were several kinemages in one file. It is a very simple matter to make a single file containing two or more kinemages.
Caution: It’s best to completely finish editing your individual kinemages before you merge them. Also, after merging, only the text window of the first kinemage will be seen but each caption window will appear with its kinemage.
To merge, open the first kinemage in a word processor and put the cursor at the end of the file. Then insert the second kinemage file into this one. In the second kinemage, delete its @text entry (not the @caption), and change the @kinemage 1 to @kinemage 2. Go back to the top of the entire file and under the @text add a table-of-contents entry for Kin 2, and add more text to explain what it shows. Save the file as plain text and rename it
Animating between views – Save a specific views of specific groups in MAGE, eg View 1 shows group X, View 2 shows group Y, View 3 shows group Z. Now open the kinemage in a word processor and next to the "@group {X}" add "animate moview= 1" . Do the same with the other groups. This tells MAGE that animation between these groups will turn on the specific views of those groups. See kinemage 4 of c2motifs.kin in the Branden and Tooze (2nd ed) series for an example.
Coloring individual ribbons – Open the kinemage in a word processor. Ribbon kinemages are more complex in format than others, but at the top of file should be several lines beginning with @colorset such as @colorset {alpha} gold . If you changed this to @colorset {alpha} red , then all of the alpha helices would show up as red instead of gold when you opened this kinemage in MAGE. To change a particular ribbon to a particular color, you will have to find the vectorlists associated with that ribbon and change the color of each of those vectorlists. You could alternatively change the color on those vectorlists to a defined term like "ribbon1" and then create a new "@colorset {ribbon1} yourcolor" at the top of the file.
Using master buttons – From demo5_4b.kin: "Master buttons provide a way of controlling display objects in sets that are independent of the group-subgroup-list heirarchy. They are specified by adding a parameter "master= {masterbuttonname}" to the first line of each of the objects; for each different masterbuttonname, the program will make a master button. Any time there are multiple groups with similar objects in each, it is probably best to use dominant groups and master buttons." PREKIN automatically adds master buttons (below the group-subgroup-list heirarchy) to most kinemages. Examining the kinemage with a word processor will show you the relationship between these two different types of buttons, and will allow you to alter them to suit your needs.
Changing
the radius of a ball or sphere – To
change the radius of an atom ball or sphere representing a metal ion, open the kinemage in
a word processor. For a sphere, there will be a line beginning @spherelist and within that
line will be radius= X, where X is some number such as 0.5. Changing X will change the
radius of the sphere, which might be desirable if you are representing metal substitutions
in a molecule.
This exercise will translate the coordinates of one PDB file to match those of another one. In this way, kinemages made with the information from one PDB file will overlay any kinemages made with the other PDB file and superimposition will be unnecessary.
Begin by downloading DeepView (SwissPDB Viewer). Go http://www.expasy.ch/spdbv/mainpage.htm , click on Download, then Accept License Agreement. On the next page select the particular version you want to download. Most will have a PC, so click on PC, then self-extracting archive. Save to a new folder, which you have named SwissPDB within your kinemage folder on your computer. Click on the file and extract to the same folder.
Now move two pdb files to the SwissPDB folder, 2AAI.pdb and 1HMW.pdb. The first is the file you used for the ricin tutorial and the second, which you will have to download from the Protein Data Bank, is of a similar plant toxin. Although very similar, the coordinate sets of these two proteins are not aligned.
Open DeepView by clicking on the spdvb icon. Close the “about” window and click on the File menu, then Open PDB File. Find 2AAI.pdb and open it. Close the “inputlog” window which pops up and you will see a graphic image of ricin. Open File again, then Open PDB File. Find 1HMW.pdb and open it. Again close the “inputlog” window and you will see two protein structures in the graphics window. Now click on the Fit menu, then Iterative Magic Fit. The dialog box that opens will indicate that the two structures will be fit together based on their alpha carbons. Each PDB file, called layers by spdvb, are listed at the bottom of the dialog box. 2AAI is listed on top, meaning it will serve as the template for fitting 1HMW. Clicking the ok button should immediately superimpose the graphic figures. To save the “adjusted” 1HMW.pdb file, go to the Window menu and open the Control Panel. A long window will open on the right. Click and hold on 2AAI, then move the check to 1HMW. This makes the 1HMW file the active one. Now go back to the File menu, click on Save -> Layer. Save the “adjusted” 1HMW file under a new name such as 1HMWnew.pdb.
To illustrate that these PDB files are now superimposed, make a kinemage from each using only the B chains (subunit 2) of each file, then append them together.