MolProbity is a web service hosted on the WWW by the Richardson Lab at Duke University. MolProbity's Main Page can be accessed directly via the server address: http://molprobity.biochem.duke.edu/ and via the Richardson Lab website http://kinemage.biochem.duke.edu/.
Notice that two additional panes have been added to the MolProbity Main Page. Options available while running MolProbity are context-sensitive. Whereas, before loading a coordinate file, you had two panes - "File Upload/Retrieval" and MolProbity information; after loading, you also have a "Suggested Tools" pane to work on the indicated coordinate file and a "Recently Generated Results" pane to manage the files in your work area.
The tools available in the "Suggested Tools" pane are also context sensitive. We will use the "Add hydrogens" option next; but one could just as well edit the PDB file here, if for instance, there were multiple identical chains in the asymmetric unit.
His42 is blindingly obvious visually or by score differences in MolProbity, but is puzzling to assign unaided, with 3 potential H-bonding groups nearby (turn to put the His ring flat and find the 3). The preferred flip state makes 2 good H-bonds (try turning off the "vdw contact" button, for a clearer view of the pale green lenses of H-bonding dots). Measure each N to O distance (just pick the 2 atoms in succession, click on the "Markers" button near the bottom right of the KiNG window to help track your picks). Animate to the other flip state. Distance to the Ile49 carbonyl O is too far (measure it) for any but a very weak H-bond, while both ring CH's produce red clashes. The electron density shows that this ring is very clearly positioned, and the N atoms in the preferred state show higher local density peaks. (You can check this in MolProbity by fetching a map from the EDS and re-opening KiNG, but we suggest you optionally do it later in Part 2 when you are working with map and model in KiNG off-line).
The Suggested Tools pane now includes the "Analyze all-atom contacts and geometry" tool as you are now working on a coordinate file with hydrogens. Select this tool, look at the choices in this next dialog-page, add "Geometry evaluation" to both graphics and chart sections, and then "Run..." with the default settings otherwise. This initiates calculation of the set of analyses requested and the spinning-SOD entertains. However, note that analysis steps are checked off as they are completed and some present links to results immediately viewable. So, if you tire of spinning-SOD, you can look at results before all of the requested set is complete. Otherwise, you'll see next the "Analyzed all-atom contacts and geometry for 1bkrH.pdb" report. From this page you can see the summary statistics or choose to view any of the requested model quality assessments. Discussed below are the items requested for 1bkrH.
The summary statistics for 1bkr show excellent Ramachandran values, but mediocre sterics and poor sidechain rotamers for this resolution range. No backbone bond lengths or angles deviate >4σ, but there are two Cβ deviations (see below) >0.25 Angstrom. The important thing, though, is not the overall scores, but the specific good or bad local regions that produce them. Click on "Multi-criterion chart". It comes up ordered by residue number. Scroll down, to see that both N- and C-terminal residues have problems (very common, even at atomic resolution). A click on the title of any column sorts the list by its values: try "Rotamer", to put the most suspect sidechains first, and note that other pink outliers are also enriched. [A misfitting typically shows up on more than one validation criterion.] Both chain termini (res 2 & 109) and the two Thr's are outliers in 2 or 3 columns. In a 100-res protein it would be plausible to have one rotamer <1% score that was valid; however, in 1bkr all 6 are in fact wrong. "Close this (chart) window"
Back on the Analysis results page, ask to view the Cβ deviation scatter plot in KiNG. Either zoom way out or choose View2 to understand the bulls-eye pattern of experimental points relative to an ideal-geometry Cβ atom. 1bkr has most points in a very reasonable distribution, but with 3 clear outliers (click on each to identify) (turn off the "bullseye" or zoom in to make picking clearer): Lys 108 is at the high-B C-terminus, and Thr 77 and Thr 101 sidechains are misfit, as you will see. [If the distribution is highly asymetric or extremely broad, then probably something was amiss with the angle restraints during refinement. Alternate conformation sidechains with common Cαs also often produce large Cβ deviations - understandable, but not ideal.] Close the KiNG window.
The multi-criterion kinemage shows the Cα backbone, with all-atom clashes as hotpink spikes, bad Cβ deviations as magenta balls, and poor rotamers as gold sidechains (Ramachandran outliers would be flagged by heavy green lines, and bad bond angles in blue or red). Again, the two Thr and the chain termini show up clearly as clusters of problems. Go to Lys 2 (either locate it visually and right click to center, or use "Find point" on the "Edit" menu) and turn on buttons for mainchain, sidechain, H's, and water rather than Cαs.
Check B-factors (click on atom and read info line at bottom of graphics window) for some non-terminal nearby Cαs as controls, which should be around 10. Then try the sidechain atoms of Lys 2; the clash with Asp 6 is probably just a misplacement of the Lys sidechain. The Lys N clashes with a water (both relatively low B); this can be well fit as the 1-2 peptide to the missing residue 1 in helical conformation (the water becomes the carbonyl O); optionally you can confirm this later by looking at the 2Fo-Fc map. The Multi-criterion kinemage contains a wealth of information, which we will explore off-line, so for now, close the KiNG window.
The bottom of the Summary statistics page shows the MolProbity statistics as a REMARK 40 for the PDB header. The WorldWide PDB has approved this remark purely as a citation for validation programs other than the official PDB ones, but has decreed that no results may be reported! Hopefully that policy will soon be altered, so that you can brag suitably on your own excellent structures.