Part 4: Rebuilding in O

done on 1SBP, a sulfate-binding protein at 1.7Å resolution.

Prepare, by checking

  1. that two programs from our lab are installed and on your PATH -- Reduce - to add hydrogens to proteins, nucleic acids, and ligands, and Probe - to generate the contact dots. From the command line of a terminal type:
    shell> reduce -help and then,
    shell> probe -help,
    to check that these programs are in your PATH.
  2. that your O installation is reasonably functional. You do need the global variable, $ODAT, defined; you don't need the symmetry operators, but they're good to have; and you do need the standard O set-up files - menu.odb, startup.odb, access.odb, and a modified version of the O distributed stereo_chem.odb.
  3. that you have two files from {TBD} in your own working directory: 1SBP.pdb (the coordinates file from The PDB) and 1sbp.omap (a 2Fo-Fc map which was calculated from the deposited structure factors and obtained as an O-map from the Uppsala EDS site).
  4. and that you have two files from the Richardson Lab: probe.mac - an O macro which coordinates O, Reduce and Probe to draw the needed O objects; this can be in the ODAT directory, OMAC (if that still works) or in the local directory (if you want to customize it); and prbsphereconv- an awk script which reformats O atom descriptions to Reduce atom descriptions, this has to be in your PATH.

Fire-up O and read in the model and map

  1. Type the command to start-up O:
    shell> ono
  2. {This item is likely to change, depending upon the particulars of the local 'O' installation.} Do not initially define an O file, e.g. just <CR> for no initial O file, then accept the default menu.odb, then default startup.odb. When the stereo chemistry database question comes up, you might be instructed to enter another database which will contain rotamers from the Richardson Lab. Finally, accept the default access.odb.
  3. Now you should have an O graphics window! Commands can now be typed into that O graphics window, or into the parent X window which can be resized and moved to show at the bottom below the O window.
  4. Read in the coordinate file, giving it a 5 character name. (Note ";" represents an O default value):
    ono> pdb_read 1SBP.pdb sbp-1 ; ;
    Make an object using everything:
    ono> mol sbp-1 zo ; end
    Center on the molecular object:
    ono> ce_zo sbp-1 1 448
  5. Read in the 2Fo-Fc map, giving it a name (up to 6 characters) like '2fo':
    ono> fm_file 1sbp.omap 2fo ;
    (Space Group is P212121 but this is not needed.)
    then use the density pull-down to colo(u)r and conto(u)r:
    density>2fo and then use the sliders. (You might try for Rad(ius) 8, Level 1.5, Red 0.2, Green 0.5, Blue 0.5 (approximately).) Turn off the Cα trace, object SBP-CA in the objects menu.
  6. Then read in the difference map, giving it a name (up to 6 characters) like 'diff':
    ono> fm_file 1sbp_diff.omap diff ;
    use the O command, fm_setup, to color and contour the difference map at two levels:
    ono> fm_setup DIFF 15.0 solid 2 -3.5 red 3.5 green

Do the set-ups for all-atom contacts

  1. Put the probe.mac macro on your user menu:
    ono> menu @probe.mac
    the '@' sign is critical here! Choose "on".
  2. If you want to do so, now would be a good time to save your O datafile:
    ono> save my1sbp.ono
    or use the Controls>Save menu item.

Check-out the model vs all-atom contact dots and electron-density map

In the previous session on MolProbity, one potential area of interest for 1sbp was the threonine residue 32. Having two sets of bad-overlap contact dots, a high Cbeta deviation, and a bad rotamer score, this area is "yelling for help". In this section, we will use all-atom contact analysis to help us rebuild that area/residue.

  1. Center on the area we'll be working on:
    ono> ce_at sbp-1 32 CB
    Zoom in, and perhaps slab down some. If needed, use the menu command Controls>Centre on atom to recenter. Also, turn off display of the maps by the Menus>Objects pulldown. It is very convenient to tear-off the Menus>Objects, the Menus>User menu, and the Menus>Fake dials menu and place them around the edges of the O window.
  2. First check-out the contact dots. Activate the Menus>User menu>probe.mac Omacro by clicking it once on the Menus>User menu pull down. Then, click once on the 32THR CB atom. You will have two new objects:
    PRBSPH - a copy of the model out to 6Å from the Cβ, and
    self_dots - the contact dots.
    Their display (as well as the map display and other O objects) can be toggled using the Menus>Objects pull down.
  3. Now check out the map. Use the Menus>Objects pull down to toggle the map on and off; likewise with the dots.

See if another rotamer helps

  1. (Optional) Construct a duplicate molecule and object to use as a comparison in what follows.
    ono> pdb_read 1SBP.pdb sbp-2 ; ;
    ono> mol sbp-2 zo ; end
    Now you can turn off SBP-1 and PRBSPH in the Users>Object menu, then Display>Paint Mol>White to set paint color and then Display>Paint Mol>Paint Object whereupon you will be prompted to pick an atom of the object to paint. Picking an atom of SBP-2 will paint the entire object white. Now, turn SBP-2 off and SBP-1 on to continue; later after changing SBP-1 (below), you can turn on SBP-2 to see a white ghost of the original.
  2. Use the Rotamer to build menu item to display and evaluate threonine rotamers for possible substitution, it is located down in Rebuild>Database>Rotamer to build
    Select the Rotamer to build menu item and then click on an atom of residue 32. O will substitute the first rotamer in the database.
    You don't want to do this command at the moment, but selecting the Controls>Yes menu item will accept the displayed rotamer and update the model while selecting Controls>No will revert back to the previous state.
  3. You can flip through the rotamer database in numerous ways: the Menus>Fake dials sliders, the keyboard arrow keys, or a dial box with selection via Controls>Dials previous and Controls>Dials next pulldowns. You may need to click in the "Dial Menu" to get it to update what items are available.
  4. Use the electron density map and contact dots to guide your selection of a new rotamer, the probe macro will work while the rotamers are active. Select Controls>Yes to accept it. (Turn on SBP-2 to compare with the original model.)

    Hint: If either you or O gets confused, as to what the dots are representing, it is best to delete the PRBSPH and self_dots objects (Display>Delete object>PRBSPH) and get a fresh look.

Improve the fit to map using real space refinement

After selecting a rotamer, we're left with a slightly poorer fit to the map. But contrariwise:, the map is biased to the original model; relocation of the OG1 helps explain the positive difference density; the original CB placement yields a high Cbeta difference; the map density for the OG1-CB-CG2 sidechain is linear, rather than lobed (cf. Thr 96) and there is a hint that this straight portion may be bent as with the proposed rotamer. With this accumulated evidence, its reasonable to think that refinement and a new map will lead to a better explanation of the data. Let's use the current map to improve the model's fit locally.

This step is optional for this particular residue rebuild when using O. As a matter of fact, refinement of the newly placed rotamer against the 2Fo map appears to worsen the placement of the threonine for this example. But note the difference in Cbeta placement between O and Coot (view the screen shot for Coot). In O, the Fast_Map commands are used for real space refinement, instructions for their use can be found on Alwyn's A-Z of O page.

  1. Choose the map to refine against from the Density>Density menu item. Choose "2FO", if it is not already selected.
  2. Activate the fm_rsr_zone command by choosing the menu item, Density>E.D. Fit>RSR zone. You will be prompted to choose two items to define the zone for refinement; for this case, click two atoms in residue 32 (same atom works).
  3. You can use probe.mac to see the contact dots for the atoms repositioned in the real space refinement before accepting the coordinates.
  4. Selecting the Controls>Yes menu item will accept the displayed positions of the residue's atoms moved in refinement and update the model while selecting Controls>No will revert back to the previous state.

Twiddle with the fit using torsion angles

Rotating the threonine's side chain oxygen round not only uses the rotamer of highest frequency, it also creates a new hydrogen bond between the amide hydrogen of ASP 34 and the THR 32 OG. With rotation of the OG1-HG1 hydroxyl bond yet another H-bond can be created between this hydroxyl hydrogen and the carbonyl oxygen of TRP 28.

While O has a good facility to adjust torsion angles and an example is shown below, O does not readily display hydrogens. So, it is suggested that these fine twiddles be done in KiNG. Still, torsion angle adjustement is shown below.

  1. Activate the Rebuild>Torsion>In Residue menu command, then select an atom of Thr 32 to be able to adjust the defined torsion angles. Adjust Chi1 as you see fit using the Menus>Fake dials sliders, the keyboard arrow keys, or dial box. Unfortunately, since the torsion command disconnects the residue, the probe macro will not work while torsions are active -- you must make a good guess, then accept with Controls>Yes, then execute the probe macro to check out the new conformation.

Congratulations, you've rebuilt a problem area.

  1. At this point, one could write out the coordinates and go to the next problem area. Controls>Save only saves the binary O file. To write out the coordinates to a text file to be used in another program, do:
    ono> pdb_write 1sbpRbld.pdb sbp-1 ; ; ; ; ;

This technique is now available to you whenever fitting or validating. It is most valuable, and easiest to apply, when done during the refinement and rebuilding process rather than waiting until the end. Use it whenever you are trying to make a difficult decision between alternatives, run the overall validation (as in the first part of this exercise) occasionally to find problems you might have overlooked, and be sure to check one last time before depositing coordinates.
Jane & Dave Richardson