Hncacb

NMR experiments for resonance assignment were performed on a Bruker Avance III HD 800-MHz spectrometer equipped with a cryogenically cooled probe head. All samples were prepared in a buffer containing 20 mM Sodium Phosphate pH 6.3, 100 mM KCl, and 5% (v/v) D₂O. The NMR spectra for backbone resonances assignments were collected using a 450 μM uniformly ¹³C, ¹⁵N–labeled DrFoxM1 TAD protein. NMR labeled TAD protein was phosphorylated by Plk1 kinase domain as described above. Sequence-specific backbone resonance assignments for DrFoxM1 TAD were determined using HSQC, HNCO, HNCACB, CBCA(CO)NH, and C(CO)NH experiments supplied by Bruker BioSpin. Sequence-specific backbone resonance assignments for phosphorylated DrFoxM1 TAD were determined using HSQC, HNCO, HNCACB, and CBCA(CO)NH experiments supplied by Bruker BioSpin.

Assignment of backbone amide peaks of Yth1 constructs was carried out using the following standard triple-resonance spectra: HNCO, HN(CA)CO, HNCA, HNCACB, HN(CO)CACB, HN(CAN)NH, and HN(COCA)NNH (Bruker). TROSY versions of these spectra were used for the backbone assignment of Fip1₂₂₆. Backbone data sets were collected with nonuniform sampling at 20%–50% and processed with compressed sensing using MddNMR package (Jaravine et al. 2008 (link)). Resonances from proline residues in Fip1₂₂₆ were assigned using ¹H start versions of ¹³C-detect CON, H(CA)CON, and H(CA)NCO (Bruker). Backbone resonances were assigned manually with the aid of Mars (Jung and Zweckstetter 2004 (link)). Topspin 3.6 (Bruker) was used for processing and NMRFAM-Sparky 1.47 (Lee et al. 2015 (link)) was used for spectra analysis.

All NMR spectra were recorded at 298 K on an 800-MHz Bruker Avance III HD spectrometer equipped with a cryo-probehead. All spectra were processed with NMRpipe [72 ] and analysed with NMRFAM-SPARKY [73 (link)]. For titrations with SIMs, standard 15N-HSQC were recorded for each protein-ligand concentration. Standard triple resonance CBCA (CO) NH, HNCACB, HNCO and HN (CA) CO experiments were used from Bruker library for backbone assignments using a ~ 1 mM uniformly ¹³C,¹⁵N-labelled NbSUMO. Following peak picking of the backbone experimental data in Sparky, the chemical shift lists were submitted to PINE NMR-server [74 (link)], and the assigned peak list was verified and completed manually. For the structure of NbSUMO, the above chemical shift lists were submitted to the CS-Rosetta server along with the primary protein sequence. From the obtained structures, the best PDB was reported out of.