Part 4: Further Exercises - now or later, try one or more of these

A. Self analysis

Choose one of your own structures, or one you're especially interested in. Analyze it, either in MolProbity, or add H's in MolProbity or in phenix.refine and analyze it in the Phenix validation GUI. Then try making some corrections in KiNG or Coot.

B. Low resolution - an H in 50S ribosome protein/RNA contact at 2.4Å

  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 the associated pdb (1s72H_d2_clowy.pdb) and map (1s72_Lccp4.map) files into your working directory. (CSHL students, these files are available in ~/dcr/).
  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 0.9σ, and try refitting this one. Or, just explore the all-atom contacts for these protein/RNA interfaces.

C. Low resolution - an Ecoli ribosome 70S protein/protein contact at 3.2Å

Get the kinemage, pdb, and mtz files for 3i1n from ~/dcr.

First look at 3i1n_L13L20-multi.kin and its 2Fo-Fc map in phenix.king, to diagnose the problem around L20 Arg 63 (there's a view, and a map). Then bring the mtz and pdb files up in Coot and see whether you can fix this entangled set of problems, starting from Arg 63. If you want to check the answer, turn to page 42... that is, to chains J & Q in PDB file 3r8s.

http://kinemage.biochem.duke.edu/teaching/workshop/MolProbity/
Jane & Dave Richardson