Title: IDENTIFYING A COMPENSATING MUTATION TO RESCUE FUNCTION IN BRUTON’S TYROSINE KINASE WITH AN XLA MUTANT Mutation model: The coordinates template of Btk linker-kinase used: PDB ID: 3K54 The N-terminus of the αC-helix is modeled using Csk (PDB ID: 1K9A) and Lck (PDB ID:3LCK) The activation loop are modeled based on Btk (PDB ID:1K2P) Mutation is modeled in MODELLER (https://salilab.org/modeller/) Molecular Dynamics Simulation: VMD (v 1.91) was used to solvate and ionize the protein molecules. It can be downloaded from (https://www.ks.uiuc.edu/Research/vmd/vmd-1.9.1/). NAMD (v2.10) was used to run the minimization, equilibration and production simulation of the proteins. It can be obtained from http://www.ks.uiuc.edu/Research/namd/. All the protein molecules were first prepared for simulation using genFiles.tcl. They were then subjected to short simulation using minEquilWater.conf and minimization.conf. Equilibration runs were carried out using equilibration.conf configuration file. The simulations were then carried out in intervals of 20ns intervals using 20ns.conf. The list of file used by NAMD: 20ns.conf minEquilWater.conf minimization.conf equilibration.conf par_all27_prot_na.inp: forcefield parameter file The trajectory files of wild type protein molecular dynamics simulation: 3K54_Linker_500ns_100ps_CA.dcd 3K54_Linker_500ns_100ps_CA.pdb The trajectory files of single mutation molecular dynamics simulation: 3K54_Linker_R641H_550ns_100ps_snapshots_CA.dcd 3K54_Linker_R641H_550ns_100ps_snapshots_CA.pdb The trajectory files of double mutations molecular dynamics simulation: 3k54_Linker_R641H_K420R_250_CA.dcd 3k54_Linker_R641H_K420R_250_CA.pdb High order dependence calculation: All the source codes for calculating SDII are available at: https://github.com/jkjium/contactGroups Dataset used in the calculation: PF07714.fa (Pfam version 30.0): Pfam multiple sequence alignment. (https://pfam.xfam.org/family/PF07714) Parameters used in this work: gaps cutoff for removed columns: 0.2 (more than 20% gaps) sequence weighting cutoff: 0.7 (less than 70% similarity) For any question please contact Kejue Jia (kjia@iastate.edu)