Part 4: Optional Exercises - try one or more of these further exercises

A. Self analysis

Choose one of your own structures, or one you're especially interested in, and analyze it in MolProbity.

B. Why Backrubs are therapeutic - a buried Ile in 1mo0

Procedurally, this example is the same as that of Thr 77 in Part 2. For Ile A120 in 1mo0, selection of the correct rotamer is difficult: the map does not indicate a clear choice and neither do probe dots. The key idea behind the Ile A120 rotamer selection is that one of the rotamers (pt) gives clashes on only one face of the sidechain. The backrub tool then allows one to finish the correction from the pt rotamer.

C. In the ribosome

  1. The file 1s72H_d2_clowy-intface.kin.gz contains about 30% of the RNA from the 50S ribosomal subunit, plus 5 of the interacting proteins (chains c,l,o,w, & y). Copy it and all the associated pdb (1s72_d2_clowy.pdb, 1s72H_d2_clowy.pdb and map ( files into your working directory.
  2. Read the .kin.gz file into KiNG, choose the view for L Arg 6, and read in the map using Tools > Structural biology > Electron density maps (note that it's in CCP4 format). Both Arg and G base fit their density well, and the guanidinium stacks nicely with the U above it (green vdW dots), but it seems to be trying, and failing, to make Arg-G H-bonds. Choose Tools > Structural biology > Sidechain rotator, select the pdb file (be sure to use the 1s72H one with H atoms, not the plain 1s72 one) and pick an Arg atom to enable fitting.
  3. Right-click on the Cb and look down the Ca-Cb bond to judge the density placement, which clearly requires a minus chi1 (putting Cg opposite the backbone CO). Go back to the standard L Arg 6 view, scroll down in the rotamer list, and quickly try each one that starts with "m", and look for one that places the guanidinium somewhere close to the right position and orientation but flipped over about 180° from the original (don't worry much about clashes at this stage). Starting from the one rotamer that's promising, adjust the chi dials to get 2 H-bonds and a good density fit. There is still a small clash when the H-bonds are good; that level is not very worrisome, but you can improve the interaction using a small backrub change if you like. Now you have a classic double-H-bond Arg-G specific interaction. Accept your refit.
  4. There are several other misfit Arg/RNA interactions in here (they understandably worked harder on the RNA than on the proteins), but only one other within the map provided. If you like, go to the C Arg 246 view, take the contour level down to about 0.9 sigma, and try refitting this one. Or, just explore the all-atom contacts for these protein/RNA interfaces.

D. Cross training

  1. Try Coot or O on the arginines in 1bkr or 1s72, or try the 1sbp Thr in KiNG.
  2. [Note that Coot needs CCP4-format maps; we've provided one for the ribosome piece, used also in KiNG. Currently Coot's rotamer function works better without hydrogens, so use the 1s72 pdb file not the 1s72H one. Once you've accepted a rotamer, then add H atoms and contact dots with Validate > Probe clashes. For some Arg (especially 1s72 C 246) you'll need to lower the rotamer probability threshold in order to get the nearest flipped rotamer to show up in Coot's list (consult the help, for how to script this). Once you find the nearest starting rotamer, though, Coot's real-space refinement does a marvelous job of homing in on the exact conformation.]
Jane & Dave Richardson