Computational protocol: Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4

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[…] SUMO dimers were dissolved in 300 μl NMR buffer containing 20 mM Tris–HCl, pH 7.0, 100 mM NaCl, 2 mM TCEP and 10% D2O to a final concentration of ~300 μM. A full set of triple resonance experiments including HNCACB, CBCACONH, HNCO and HN(CA)CO were recorded for backbone assignments, whereas HBHACONH, HCC(CO)NH-TOCSY, HC(C)H-TOCSY and (H)CCH-TOCSY were recorded for side-chain assignments. Peaks picked from 3D 15N/13C-edited NOESY spectra together with dihedral angle restraints derived from TALOS+ were used in ARIA to calculate the SUMO structures in the dimers. Similar procedures were followed after formation of complexes between SUMO dimers and the SIM peptides, to assign chemical shifts and calculate the SUMO structures in the complex environment. Combined chemical shift perturbations were calculated using [(1H difference)2+((15N difference)1/5))2]0.5. [...] 15N T1, T2 parameters were measured for both SUMO-2 domains in free and complex states. The model-free approach was used to derive rotational correlation times for each data set. For spin labelling, the free cysteine of the SIM peptide was covalently bonded to MTSL. An amount of 1.7 mg of peptide was dissolved in 450 μl buffer and 5 mg of spin label was subsequently added in 50 μl DMSO solution. The mixture was incubated overnight at 277 K and washed using a concentrator to remove unreacted reagents. 1H–15N HSQC spectra of segmentally labelled SUMO-2 dimer were recorded in the presence of saturating amounts of spin-labelled SIM peptide. The paramagnetic effect was removed by reduction with ascorbic acid, and a final HSQC recorded. RDCs were measured using In-phase Anti-phase (IPAP) experiments with samples containing 14 mg ml−1 Pf1 phage. The alignment tensor was obtained using PALES and then used in CNS for modelling of the complex structure. […]

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