Part 1: MolProbity Analysis
A. Analyze 1BKR and prepare it for rebuilding in KiNG.
Fire-up MolProbity and read in the model
MolProbity is a web service hosted by the Richardson Lab at Duke University. MolProbity's Main Page can be accessed directly via the server address: http://molprobity.biochem.duke.edu/
There is no set-up required for MolProbity - it will run in a "modern, java-enabled" browser. Safari is being used as the example here. If you are using Mac OS X, please use either Safari or Firefox. Java is enabled via the browser preferences set-up. More detail about Java (especially needed for Windows) is available from the Installing Java link off of the MolProbity Main Page.
Enter "1bkr" in the field for choosing a PDB/NDB code and click "Fetch >". Note the molecule identity (CH domain), resolution (1.1Å), number of chains/residues (1/108), crystallographic details, etc. in the resulting info panel, and rotate the small Java thumbnail. [This lets you see if you mistyped the PDB code (e.g. 0 vs o) or if there are more molecules in the asymmetric unit than you wanted, etc.] Click the "Continue >" button to continue to the MolProbity main page.
Add hydrogens & evaluate Asn/Gln/His flips
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.
Choose the "Add hydrogens" function, and accept the defaults on the next dialog-page: "Start adding H >", to run Reduce with Asn/Gln/His flips and electron-cloud x-H distances.
All suggested flips are for His (no Asn or Gln) and seem like clear wins from the scores. Choose "View in KiNG" for the 1bkrH-fliphis.kin file ("his", not "nq"). The KiNG "Views" pulldown menu has an entry for each His, with * marking those flipped by Reduce; look at each * view. H25, H73, and H104 are clear and simple; rotate the viewpoint to see the H-bond partner(s), and to compare the two flip states use the "a" key or the Animate arrows in the lower right side panel. [Side chain is colored green in the preferred state.]
His 73, as shown definitively by contacts, does indeed need to be flipped. Note that both orientations are protonated at the NE2 position, but in the flipped orientation, the ND1 atom could potentially also be protonated and donate to the nearby water. Such protonation difference doesn't affect R factors, but the change in charge would destroy an MD or docking calculation; one has to really examine this case to see if double protonation would be more reasonable. Upon closer examination, the water nearest ND1 H-bonds to Arg 45. Arg is charged and an obligate donor, so this water is not likely to favorably receive another donated H from the His. Hence, His 73 is probably accepting an H from the water, and thus would be singly protonated on its NE2. It is important to note that the program Reduce, which adds hydrogens and optimizes N/Q/H orientations in MolProbity, does not take the Arg 45 into account when determining protonation state, and if the inter-atomic distances were slightly different, could double-protonate His 73. This emphasizes that there is no substitute for a person really looking, evaluating, and possibly making further changes as a model is constructed.
The correct flip for 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" and "small overlap" buttons, 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. The distance from the His42 ND1 to the Ile49 carbonyl O is too far (measure it) for anything 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).
Close the KiNG window (button at bottom of page). You now have the choice of rejecting a flip if you don't agree with it. [That's rare, but can happen, especially if you have access to extra information. For example, if a flip state is completely unambiguous in one crystal form (e.g. with ligand bound), then "some evidence" is probably not enough to justify fitting it differently in another crystal form.] These 4 His flips in 1bkr should very clearly all be accepted, so just click the "Regenerate H,..." button, which moves you on to a flip-report page. Note the information presented on this report and then "Continue >" to the MolProbity Main Page.
Analyze all-atom contacts and geometry
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.
Summary Statistics & Multi-criterion chart
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. "Continue" back to the main page.
Download files for use off-line in Part 2
"Continue" back to the main page. Expand "coordinates" in the file download section (by clicking the little triangle), find the pdb file WITH the new H atoms: 1bkrFH.pdb, and download it to your working directory for this practical (best to right-click and "save link as"). Now expand "kinemages" and download 1bkrFH-multi.kin.gz.
Log out (on left side navigation panel), and "destroy" all files. (Note in file names: F for Flipped, H for Hydrogens added.)
Go to the EDS (http://eds.bmc.uu.se) Electron Density Server and type in 1bkr. Choose "Maps" on the lefthand list, ask for 2mFo-DFc in O format, and download the result (right-click). Ask for mFo-DFc, download, and exit the browser. You should now have a PDB file with H atoms, a multi-kin, and two map files for 1bkr in your working directory. [The last three files are compressed as *.gz, but you won't need to unzip them.]
Now you have the files to continue working off-line with KiNG to rebuild selected residues in Part 2...
Jane & Dave Richardson