Computational protocol: Structural analysis on mutation residues and interfacial water molecules for human TIM disease understanding

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Protocol publication

[…] As the sequences of the wild type HsTIM and E104D mutant differ only from each other in one position, we perform structural alignment of the two structures by superimposing them using Pymol []. The algorithm is based on the amino acid sequences of the two structures. Once the aligned wild type and mutant structures are obtained, the 25 water molecules in wild type are searched in the mutant structure to determine whether it reappears or not. If two interfacial water molecules, one in wild type and the other in mutant, are within 1.0 Å distance from each other, and they are mutually the closest water molecule to each other, we say the water in the wild type structure reappears in the mutant.Due to the environmental differences when a protein structure is solved, the B factors in different structures cannot be compared directly. The B factor of a protein complex is normalized within the complex as: (3) B * = B - B ¯ a σ a where B is the original B factor reported in the PDB record, and B ¯a and σa are the mean and standard deviation of the B factors of non water atoms which are at least 15 Å away from the Cγ of the mutated residue--GLU104. We do not consider water here because the water information quality is correlated with the resolution [] and, maybe, other environmental issues. We also exclude the atoms near the mutated site from calculating mean and standard deviation as the mutation is expected to change the B factors of atoms near it. In this way we compare the B factor of an atom in wild type and mutant with non water atoms far from the mutation site as references. [...] Two molecular dynamics simulations were run for the wild type [PDB: 2JK2] and the E104D mutant [PDB: 2VOM] HsTIMs. Buried water molecules were included in the initial structures. For both simulations, the protein structure was solvated in a water box where a minimum of 10 Å distance was kept between the protein and the boundary. Charges were neutralized by either Cl- or Na+ ions. Solvation and ionization were performed using the the VMD software [].For both simulations, CHARMM22 parameter set [] with CMAP correction [] was used, and the step size was set to 2 fs. The solvated and ionized system was minimized for 1000 steps and simulated for 2500000 steps (5 ns). Initial velocities were set with human body temperature (310 K). Langevin piston pressure control was used to control the system pressure at 1 atm. Periodic boundary conditions were used, and a threshold cutoff of 12 Å was set for non-bonded interactions. Particle mesh Ewald method [] was used to calculate long-range electrostatic interactions. The simulations was carried out with the NAMD software []. […]

Pipeline specifications

Software tools PyMOL, VMD, CHARMM, NAMD
Application Protein structure analysis
Organisms Homo sapiens
Diseases Genetic Diseases, Inborn