Multiple (N) Point Mutation DDG

Calculatiung Free Energy cCHange of Multiple Mutations with Bench

a 2 Mutation Example, the DDG of (D20N, V30I) in 2L32

  1. Run Prepare and call the resulting structure “Native”
  2. Create a selection of Mutant Residues (20 and 30) a cartoon of 2L32 with residues 20 and 30 in balls
  3. Use the Design Residue Selection to Constrain Design to all Desired Mutations, (e.g., D20N and V30I) a screenshot of the Bench design selector with D20N and V30I selected
  4. Run 10 Repetitions of Design and Select the Structure with the Lowest Score. Let’s call it “Mutant”
    a screenshot of Bench with 10 structures generated
  5. Repack + Minimize Step
    1. Starting from “Native”, Run 50 Repacks, then select all output structures and Run Minimize
    2. Starting from “Mutant”, Run 50 Repacks, then select all output structures and Run Minimize\ cartoons of 3 minimized, repacked, and aligned structures
  6. Sort and Select Minimize Results
    1. Sort the “Native” Minimize Results and Select the 5 Lowest Scores
    2. Sort the “Mutant” Minimize Results and Select the 5 Lowest Scores
  7. Calculate DDG, e.g. DDG (D20N,V30I)
    1. Download CSVs of Lowest 5 “Native” and “Mutant” Scores
    2. Calculate Mean of Lowest 5 “Native” Scores. Let’s call it “Native” Score
    3. Calculate Mean of Lowest 5 “Mutant” Scores. Let’s call is “Mutant” Score
    4. DDG = Mutant Score – Native Score

A negative DDG score indicates favorable mutations

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