[Kinemage Web Site]

Structure Validation Workshop

Using All-Atom Contact Analysis for Model Diagnosis & Repair

[MolProbity Server]

Part 3: Local backbone adjustments using KiNG

(done on file 1mo0).

Not all disallowed contacts can be resolved by rotamer selection and sidechain dihedral adjustments; sometimes, local backbone adjustments are needed to sample a broader space. This lesson looks at a set of clashes about Ile120 of 1mo0, a C. elegans triose phosphate isomerase. We use KiNG - a Java-encoded kinemage viewer - to select an appropriate rotamer and adjust the mainchain and sidechain.

Survey the problem

  1. The files 1mo0H.pdb, 1mo0H-contact.kin and 1mo0.omap.tgz are available on the software CDs and are probably in your working directory already; if not, add them. The first two were produced in MolProbity, and the 1mo0 2Fo-Fc map was previously downloaded from the EDS site (http://fsrv1.bmc.uu.se/eds/).
  2. In your working directory, start KiNG, and open the 1mo0H-contact.kin file from within KiNG. (KiNG keeps its own current directory so it can find all of its helper programs; in the setup here you will need to navigate up and back down again to your own working directory each time you open a file from KiNG.
  3. Lower the display overhead on your computer by clicking off components of the kinemage (your choice, but try it without wide & close contacts, small overlap and others).
  4. Center the display on Ile A120 by using the Edit>Find point... menu choice. Adjust zoom and slab to inspect clashes about Ile A120. (zoom with right-click + vertical mouse drag; slab with right-click + horizontal drag; translate in Z with middle click + horizontal drag in the upper region of the display)
  5. Load the 2Fo-Fc electron density map via the Tools>Structural biology >Electron density maps menu item.

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Try rotamers

  1. The rotamer library is invoked in KiNG via the menu item: Tools >Structural biology>Sidechain rotator. When invoking this tool, another tool, the Model manager, will prompt you to open the PDB file for your model. Answer that query (1mo0H.pdb) and a further question about alternate conformations (answer 'A' this time!) and move on by ID'ing the residue of interest (middle click on any side-chain atom of Ile A120). A pop-up window will appear allowing sidechain dihedral adjustments and rotamer selection.
  2. To update contact dots automatically as you adjust the sidechain, check the 'Probe dots' box in the Model manager window. Turn off the old 'dots' button in KiNG's side panel.
  3. Try the available rotamers to see if any look promising to alleviate the clashes seen for the residue. Probe takes a second or two to update the contacts.
  4. Pay particular attention to the "pt" rotamer, which shows a high degree of clashes, but note that the clashes are all on one face of the residue. This is a key observation leading to resolution of the clashes.

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Chiropraxis: the backrub

  1. Since all clashes are to one face of the residue in the pt rotamer, if we could rotate the entire sidechain away from that face, then we might get to a position from which we can eliminate the clashes. Such a motion requires movement of the backbone to swing the Cbeta. KiNG's backbone tool allows for such "chiropraxis" of the backbone by rotation about the axis between Ca(i-1) and Ca(1+1), called a "backrub", plus rotation of the two individual peptides.
  2. To rebuild this trouble spot in the 1mo0 model press the "Backrub" button in the rotation dialog box. Uncheck the box for idealizing the flanking sidechains (if they already fit their density well, it's better to keep them as is). Turn on the probe dots and/or ED map and adjust the pt rotamer to remove the clashes, keep fairly good geometry and satisfy the ED map; mainly use the C-alpha hinge tool, but also you can fine-tune the rotamer chi angles.
  3. Measure phi, psi, tau, chi1 and chi2 of your new Ile A120 (use the Tools>Measure angle & dihedral tool). Compare these measures to those of Ile B120 (get there with Edit>Find point..., or the Views menu).

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Save your new model

When you have resolved the clashes to your satisfaction, accept your new Ile A120 model: 'Release' the checked out residue from both tools and accept. If desired, save the modified pdb file in the Model Manager.

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Jane & Dave Richardson
previous: Part 2