Methods

Protein Structure Prediction

Several protein structure prediction programs were used to predict the tertiary structure of the Tph RT. Two different versions of the protein were submitted for folding. One sequence contained the potential additional 50 amino acids on the N-terminus of the protein, and the other sequence did not.

The protein structure prediction programs used were:

I-Tasser:

• J Yang, R Yan, A Roy, D Xu, J Poisson, Y Zhang. The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12: 7-8 (2015).

• A Roy, A Kucukural, Y Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5: 725-738 (2010)

• Y Zhang. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, vol 9, 40 (2008).

RaptorX:

• Sheng Wang, Wei Li, Shiwang Liu, and Jinbo Xu. RaptorX-Property: a web server for protein structure property prediction. Nucleic Acids Research. 2016.

• Sheng Wang, Jianzhu Ma, and Jinbo Xu. AUCpreD: proteome-level protein disorder prediction by AUC-maximized deep convolutional neural fields. ECCB 2016. Also appears at Bioinformatics 2016 (32):i672-i679.

• Sheng Wang, Siqi Sun, and Jinbo Xu. AUC-Maximized Deep Convolutional Neural Fields for Protein Sequence Labeling. ECML/PKDD 2016. Also appears at Machine Learning and Knowledge Discovery in Databases 2016 pp 1-16.

• Sheng Wang, Jian Peng, Jianzhu Ma, and Jinbo Xu. Protein secondary structure prediction using deep convolutional neural fields. Scientific Report. 2016 Jan;6:18962.

Phyre2:

• The Phyre2 web portal for protein modeling, prediction and analysis Kelley LA et al. Nature Protocols 10, 845-858 (2015)

HHpred:

• A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core. Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, Gabler F, Söding J, Lupas AN, Alva V. J Mol Biol. 2018 Jul 20. S0022-2836(17)30587-9.

• Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Gabler F, Nam SZ, Till S, Mirdita M, Steinegger M, Söding J, Lupas AN, Alva V. Curr Protoc Bioinformatics. 2020 Dec;72(1):e108. doi: 10.1002/cpbi.108.

SWISS-model:

• Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296-W303 (2018).

• Bienert, S., Waterhouse, A., de Beer, T.A.P., Tauriello, G., Studer, G., Bordoli, L., Schwede, T. The SWISS-MODEL Repository - new features and functionality. Nucleic Acids Res. 45, D313-D319 (2017).

• Guex, N., Peitsch, M.C., Schwede, T. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis 30, S162-S173 (2009).

• Studer, G., Tauriello, G., Bienert, S., Biasini, M., Johner, N., Schwede, T. ProMod3 - A versatile homology modelling toolbox. PLOS Comp. Biol. 17(1), e1008667 (2021).

• Studer, G., Rempfer, C., Waterhouse, A.M., Gumienny, R., Haas, J., Schwede, T. QMEANDisCo - distance constraints applied on model quality estimation. Bioinformatics 36, 1765-1771 (2020).

• Studer, G., Biasini, M., Schwede, T. Assessing the local structural quality of transmembrane protein models using statistical potentials (QMEANBrane), Bioinformatics 30, i505–i511 (2014).

• Benkert, P., Biasini, M., Schwede, T. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27, 343-350 (2011).

• Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L., Schwede, T. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Scientific Reports 7 (2017).

• Mariani, V., Biasini, M., Barbato, A., Schwede, T. lDDT: a local superposition-free score for comparing protein structures and models using distance difference tests. Bioinformatics 29, 2722-2728 (2013).

AlphaFold2:

Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

Structural Comparisons and Visualizations

Pymol was used to open structure data files, prepare figures, and generate electrostatic surfaces for the models.

The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

Identification of Related Proteins

BLAST was used to identify proteins with similar sequences for sequence conservation analysis.

ayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, Connor R, Funk K, Kelly C, Kim S, Madej T, Marchler-Bauer A, Lanczycki C, Lathrop S, Lu Z, Thibaud-Nissen F, Murphy T, Phan L, Skripchenko Y, Tse T, Wang J, Williams R, Trawick BW, Pruitt KD, Sherry ST. Database resources of the national center for biotechnology information. Nucleic Acids Res. 2022 Jan 7;50(D1):D20-D26. doi: 10.1093/nar/gkab1112. PMID: 34850941; PMCID: PMC8728269.