The following spectra were used for structure calculation: 3D 15N-resolved [1H,1H] NOESY (Tm = 70 ms, 800 MHz 1H Larmor frequency), 3D 13C-resolved [1H,1H] NOESY (Tm = 70 ms, 800 MHz 1H Larmor frequency), and 2D [1H,1H] NOESY (Tm = 70 ms, 100% D2O solution, 500 MHz 1H Larmor frequency). Automated NOESY peak picking and NOE assignment were carried out using ATNOS/CANDID (automated NOESY peak picking/combined automated NOE assignment and structure determination module) (36). A total of 2709 unambiguous NOESY-derived distance restraints along with 110 dihedral angle restraints derived from 13C-chemical shifts were used in the initial structure calculations performed using CYANA (combined assignment and dynamics algorithm for NMR applications). Final energy minimization and structure refinement were performed in explicit solvent using CNS 1.3 (Crystallography and NMR system), along with the RECOORD (Recalculated Coordinates Database) script package. A total of 200 structures were generated, and the 20 conformers with the lowest restraint violation energies were selected as the final representative model. The quality of the structures was assessed by the programs WHATCHECK, AQUA (Analyzing the Quality), NMR-PROCHECK, and MOLMOL. Ramachandran analysis showed that the final bundle of 20 lowest-energy conformers of GMCBM21 has excellent stereochemistry, with 97.8% of residues in the most favored and allowed region, 2.0% in the generously allowed region, and 0.2% in the disallowed region.

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