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Instructions for designing mutations to BglB using the protein editor Foldit Standalone

Update 22 Feb 2017: changed instructions to use Foldit Standalone.

Loading and saving structures

To load your protein:

"Load FASTA/PDB" from the first screen opens a file selection window, select all 5 files:

  • bglb.pdb
  • pNPG.params
  • pNPG.conf.pdb
  • BglB-pNPG.cnstr
  • BglB-pNPG.puzzle_setup

and choose "Open" to load the BglB-pNPG model into Foldit Standalone. Within Foldit, Ctrl-Alt-Shift-A to load in a structure.

To save a PDB file (XYZ coordinates): Ctrl-Alt-Shift-S.

Recommended view options

  • "Cartoon Thin"
  • "Score/Hydro+CPK"
  • show/hide bonds/voids as needed for context

Inspect individual residue energies

Mouse over residue so it is highlighted and hit Tab.

This info panel gives a breakdown of the various score terms for the selected residue. Importance of score is in order as it appears in the menu

Viewing operations

  • Zoom to Area of Interest: Mouse over residue of interest so it is highlighted; hit Shift-Q (no click) I often move around until I see the ligand and then do this

  • BackShading: Ctrl-Shift-Click (on black backdrop) and drag

  • Front Clipping: Ctrl-Alt-Click (on black backdrop) and drag

  • Measuring: Mouse over atom1 of interest, hit Ctrl-Alt-Shift-C (no click), repeat for atom 2 (reports distance), atom 3 (angle), and atom 4 (dihedral)

Selection operations

  • Select a sphere of residues around a residue that you are hovering over: Ctrl-Shift-click on residue and drag outwards

  • See upper left corner of Foldit for number of residues selected

Using the Lua terminal

  • From main options, select "Script Terminal"
  • Select specific residue: selection.Select(i), where i is the residue index
  • Zoom to specific residue: ui.CenterViewport(i)

How to design mutants in Foldit

  • Click residue and hit mutate (m) to see options for new residues
  • Selecting a subset of residues (lower right boxes) can be used to do directed designs
  • Selection will be used in both mutate and shake (i.e. shake now mutates those residues while everything else stays native).
  • Never wiggle (w) without selecting a sub-selection
  • Sometimes high energies (mutations/conformations) are need to get to the low energy
  • Native amino acid or close to native amino acid is generally better
  • Manually force the interaction you want and then side-chain wiggle (E) those residues and the ones around it
  • General order of operations: E → S → E
  • If score is not dropping faster than 0.05/s you are likely at an energy minimum. This is true only for wiggling (w/e). If you are shaking (s) pay attention to the number of cycles on the upper left corner. Often the low energy is found after 5-10 cycles (cycles is the number in the parentheses after the timer).

How to choose mutants

For an individual residue:

  • Total energy less than 0 is good
  • Total energy between 0 and +2 can be OK if clash score (fa_rep) is <1
  • Total energy between +2 and +5 are iffy, but can work if you think the Rosetta picture of the protein is missing an enabling feature
  • Total energy between +5 and +10 are are unlikely. Something will likely have to move for this to work, thus the protein is unlikely to look like your model
  • Energies >+10 are are really unlikely to work… but go for it if you have a good reason

Don't worry about the total protein score too much, as long as it is less than native, or not more than 5 Rosetta Energy Units higher than native.

Focus on the molecular interactions in the neighborhood of the residue you are mutating