NMR-Based Structure Determination of FOG-PR

Resonance assignments were made using a standard set of triple resonance experiments and NOE data were obtained from ¹³C-NOESY-HSQC (in >99% ²H₂O) and ¹⁵N-NOESY-HSQC spectra. All NMR spectra were recorded at 298 K on a Bruker Avance 600 MHz spectrometers, processed using TOPSPIN and analyzed using Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco). ¹⁵N backbone relaxation experiments (T₁, T₂ and heteronuclear NOE) were performed using standard Bruker pulse programs and were analyzed to extract relaxation rates using Sparky. Backbone φ and ψ dihedral angle restraints were derived from the assigned backbone chemical shifts using TALOS+ [47] (link). Automated NOE assignment and structure calculations were carried out using CYANA [32] (link) and the lowest energy structures were refined using the RECOORD protocol [33] (link). The 20 conformers with the lowest energy were used to represent the solution structure of FOG-PR and deposited in the Protein Data Bank (PDB accession number 2mpl). Geometrical properties were assessed using PROCHECK_NMR [48] (link).