2.4. In silico protein structural and molecular dynamic analysis

The evolutionary conservatism of all amino acids affected by missense variants was analysed using MEGA7 (http://www.megasoftware.net/previousVersions.php) with default parameters.

Protein modelling was conducted using Modeller 9V17 software 18 with default parameters based on the structure models of homozygous sequences indexed in PDB database (http://www.rcsb.org/). Molecular dynamic analysis was conducted on 1 novel missense variant. Briefly, both wild‐type and mutant models were generated by Modeller 9V17; the programme CHARMM22 was used to add hydrogen atoms, N‐ and C‐terminal patches to the models. 19 The models were solvated and neutralized in a box with TIP3P water at a minimum of 13 Å between the model and the wall of the box. All simulations were run using NAMD 2.9 with periodic boundary conditions (PBC) applied. 20 The temperature was held at 300 K while the pressure was controlled at 1 atm. The time step was set to 2 fs and the particle mesh Ewald method was applied to model the electrostatics and the van der Waals interactions cutoff was set at 12 Å. Both models followed a three‐step pre‐equilibration totalling 600 ps, the last snapshots of which were chosen as the starting structures for 20 ns productive simulations without constraints.

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