## 2 Materials and Methods  
### Modeling of the 3D Molecular Structures  
Structures of the catalytic domains of Glt1, Glt3, (GenBank codes: AAG04552, AAN67147, respectively), and Glt20 (GenBank code: KU043394) were modeled from their corresponding amino acid sequences by homology modeling using YASARA v15.10.18 software [15]. A hybrid model approach was used (parameters for the prediction are listed in Supplementary Table S1). The crystal structure of _Rm_Bgt17A (PDB code 4 WTP) [14] was the main template, while other crystallographic structures contributed with minor fragments in the hybrid models, as summarized in Supplementary Table S2. YASARA fully and automatically takes all of the steps to get the 3D model from the amino acid sequences and a detailed description of every step is available on the website.  
Firstly, all possible templates were identified by running 10 PSI-BLAST iterations to extract a position specific scoring matrix (PSSM) from UniRef90 and searching the PDB for a match. The best template structures used were selected based on the BLAST alignment score, the WHAT_CHECK score in the PDBFinder2 database and the target coverage. Secondary structures were predicted to aid alignment corrections and loop modeling. Predictions were obtained by creating a profile of related UniRef90 sequences by running PSI-BLAST, using the PSI-Pred secondary structure prediction algorithm.  
Initial homology models were built by aligning the target sequences with the available templates. These alignments considered structural information of the templates, as well as the predicted secondary structures and the structure-based alignment corrections were partly based on SSLAN scoring matrices. An indexed version of PDB was used to determine the optimal anchor points and to collect possible loop conformations. Sidechains were initially constructed as graphs and then the initial rotamer solution was determined in the context of a simple repulsive energy function. Loops were optimized by trying different conformations followed by side chain re-optimization. Sidechain rotamers were fine-tuned by steepest descent and simulated annealing energy minimization (EM) in dihedral angle space, considering packing, electrostatic interactions and solvation effects, using the YASARA2 force field. Next, unrestrained refinement of the full model was performed by energy minimization with explicit solvent molecules (water). All calculation tasks were applied to all combinations of templates and alignments giving a set of different homology models for each enzyme. Thus, 38 models were built for Glt1, 47 for Glt3 and 26 for Glt20. Every model was ranked based on the \(z\)-score, which describes how many standard deviations the model quality is away from the average high-resolution X-ray structure (negative values indicate low quality with respect to the high-resolution X-ray structure). The overall \(z\)-score is a weighted average of individual \(z\)-scores capturing correctness of backbone, side-chain dihedrals and packing interactions (overall \(z\)-score = 0.145 \(\times\) Dihedrals + 0.390 \(\times\) Packing1D + 0.465 \(\times\) Packing 3D). Finally, for each enzyme, the best parts of the respective model were combined resulting in a hybrid model with a better \(z\)-score.