<p>First and foremost, we did protein structure preparation, which is the most important aspect of in silico analysis. High-resolution X-ray crystallography-resolved structures were selected from the RCSB Protein Database. For instance, the 0.93 Å structure of Phanerodontia chrysosporium Magnesium Peroxidase (PDB ID:3M5Q) was used in one of our analyses to ensure the validity of our GROMACS molecular dynamics simulation.</p>
<p>Apart from protein structures obtained from the RCSB Protein Database, the protein structure of a newly characterized Trametes versicolor lignin peroxidase isozyme (LPG3) was prepared with ColabFold, a multiple sequence alignment (MSA)-mediated AlphaFold protein structure prediction tool <ahref="https://doi.org/10.1038/s41592-022-01488-1"><sup>[3]</sup></a>. Cofactors were reconstituted into the protein structure by AlphaFill, a homology-based ligand transplantation algorithm <ahref="https://doi.org/10.1038/s41592-022-01685-y"><sup>[2]</sup></a>.</p>
<p>MB-docked structures were prepared with SwissDock and analysed with UCSF Chimera prior to GROMACS Molecular Dynamics Simulation, for which the simulation results are shown below.</p>
<p>Our in silico analyses have demonstrated the MB degradation potential of all proteins of interest as suggested by complex formation with MB. Additionally, we plan to perform further analyses on energetics with Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) to further decouple the energetics within the system.</p>
<p>Our in silico analyses have demonstrated the MB degradation potential of all proteins of interest as suggested by our data on protein-ligand binding and interaction modes, supporting in vivo ability to form complexes with MB. This has prompted our plans to perform further analyses, such as with Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) to further decouple the energetics within the system, or co-incubation and validation with cryo-EM in wet lab.</p>
<p>Overall, the selection of all 5 proteins as potential candidates to proceed to in vitro analysis was based on literature review and our in-silico analysis workflow.</p>
<p>The idea of building a Synechococcus elongatus shuttle vector first began with iGEM Team Marburg 2019’s shuttle vector BBa_K3228089. This plasmid backbone was further modified by Team HK_SSC 2021 to create BBa_K3776009. Our team has now designed our plasmid based on these two previous contributions in view of their modularity and compatibility with our selected chassis Synechococcus elongatus UTEX 2973.</p>
