*4.5. Molecular Dynamics Simulations*

The best 18 structures resulting from energy minimization of the PONDEROSA C/S modeling were clustered by the PDB validation server (URL: www.wwpdb.org, accessed on 3 March 2021) into one 11-structure, two 2-structure and three 1-structure clusters. The best structure from each cluster was selected and subjected to MD simulations. Six MD simulations lasting 200 ns were performed using NAMD simulation software [51]. TIP3P water molecules (Jorgensen, 1983) and ions, to reach a 0.150 M ionic concentration, were added using the solvate module of the program VMD [54]. The simulation box was on average ca. 260,000 Å<sup>3</sup> and the average number of atoms was 25,554. Molecular interactions were described by amber99sb-ildn force field [55]. Protein atoms were placed at the center of a cubic box at a minimum distance of 12 Å from the edge of the box. We used Periodic Boundary Conditions set by the size of the box. The solvated systems were energy minimized by 2000 steepest descent minimization steps. The equilibration phase was performed by increasing gradually the temperature from 0 to 310 K in 100 ps followed by further 900 ps. At this stage temperature was controlled by a simple velocity rescaling procedure and pressure at 1 atm was controlled by a pressure Langevin piston [56,57], with the period of 200.0 fs and decay constant of 100 fs. The time step was 1 fs, bonded interactions were computed every 1 fs and non-bonded interactions every 2 fs. Finally, MD simulation lasted 200 ns at constant pressure and temperature, the latter controlled through Langevin dynamics with damping constant of 1 ps–1. Snapshots were collected every 1 ns along the trajectory, giving a total of 200 snapshots which have been used in the analysis.

A total of 200 structures obtained from each MD simulation at 1ns time interval were analyzed as an ensemble of structures. The RMSD from the initial energy minimized structure was obtained by superimposing the backbone atoms of the residues structured in beta sheet based on multiple alignment of annotated sequences, i.e., residues 3–7, 10–12, 18–27, 34–39, 46–51, 55–60, 68–73, 78–83, 92–98. The time evolution of RMSD during the simulation was computed in the same way. From all pairwise snapshots superpositions, the root mean square fluctuations (RMSFs) for the backbone atoms of each residue were computed. The comparison between different simulations was performed by considering the ensemble of structures from each simulation and the ensemble obtained joining the two ensembles. A large increase in RMSF upon joining the two ensembles, compared

to RMSFs observed in both ensembles, is indicative of local fluctuations about different conformations, i.e., the two simulations are sampling a different conformational space.

**Supplementary Materials:** The following supplementary material is available online. Supplementary information (Validation, Energy minimization, Assignment and Structure data); Table S1: H-bond list; Table S2: Pairwise RMSD; Figure S1: CD spectroscopy of Nb23 in H2O; Figure S2: CD spectroscopy of Nb23 in H2O with TCEP; Figure S3: ANSURR assessment for the CS-Rosetta and the NOE-restrained best cluster; Figure S4: RMSF at each residue of Nb23 in the MD simulation.

**Author Contributions:** Conceptualization: G.E., M.P., J.S. and F.F.; Methodology: G.E., M.P., Y.H. and F.F.; Formal analysis: M.P., F.F. and G.E.; Investigation: M.P., Y.H., F.F.and G.E.; Resources: G.E., M.P. and Y.H.; Writing—Original Draft Preparation: M.P.; Writing—Review & Editing: M.P., G.E., F.F., Y.H. and J.S.; Supervision: G.E., F.F.and Y.H.; Project Administration: M.P., G.E., Y.H. and F.F.; Funding Acquisition: G.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NYUAD (grant No. 76 71260 ADHPG VP046).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are openly available in the Biological Magnetic Resonance Bank (BMRB), accession number 50808, and in the Protein Data Bank (PDB), PDB ID 7EH3.

**Acknowledgments:** We thank the Core Technology Platform (CTP) of NYUAD for the access and the support with the instrumentation that was essential to achieve the reported result. To the best of our knowledge, this is the first protein structure determined in the UAE using only NMR spectroscopy. We also thank N.J. Fowler and Makek A. for the help with ANSURR software.

**Conflicts of Interest:** The authors declare no conflict of interest. The funder had no role in the design of the project, in the collection, analysis and interpretation of the data, in the writing of the paper, or in the decision to publish the results.

**Sample Availability:** Samples of the compounds are available from the authors.
