Notice that two additional panes have been added to the MolProbity Main Page. Options available while running MolProbity are context-sensitive. Before loading a coordinate file, you had two panes - "File Upload/Retrieval" and MolProbity information; after loading "1bkr", you now 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 above the original two.
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 and you wanted to focus on just one.
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, and then "Run..." with the default settings. NOTE: If your file is extraordinarily large, the default option "Create html version of multi-chart" is turned off. The run initiates calculation of the set of analyses requested. 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 the spinning-SOD ribbon, 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 1bkrFH.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 1bkrFH.
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.
Log out (on left side navigation panel), and "destroy" all files. (Note in file names: F for Flipped, H for Hydrogens added.)
Now you have the files to continue working off-line with KiNG to rebuild selected residues in Part 2...