1H-15N-HSQC spectra of 15N-labeled samples were recorded in 50 mM Tris pH 7.4, 150 mM NaCl, 200 μM ZnCl2, 1 mM DTT, 10% D2O at 25 °C with a Bruker 600 MHz Advance III spectrometer equipped with a TCI CryoProbe (Unitat de RMN, Universitat de Barcelona). NMR spectra were processed using Bruker TopSpin 3.0 and analyzed using CcpNmr48 (link). Combined chemical shift differences were calculated using the following equation:
CSP = in which α is 0.14, but 0.2 for Gly49 (link). For backbone assignment of 13C15N-RNF125start31/stop129 in 50 mM Tris pH 7.0, 50 mM NaCl, 200 μM ZnCl2, 1 mM DTT, a set of standard triple resonance experiments for the sequential assignment was recorded using incremental non-uniform sampling50 (link) on Bruker 800 MHz Avance III HD spectrometer equipped with 5 mm TCI CryoProbe (Swedish NMR Centre) at 25 °C. For 13C15N-UbcH5a assignment in 50 mM Tris pH 7.0, 150 mM NaCl, 200 μM ZnCl2, 1 mM DTT, recording was performed with a Bruker 900 MHz Avance III HD spectrometer equipped with 5 mm TCI CryoProbe (Swedish NMR Centre) at 25 °C. Spectra were processed using MDDNMR51 (link) and the analysis was performed in CcpNmr Analysis.
CSP = in which α is 0.14, but 0.2 for Gly49 (link). For backbone assignment of 13C15N-RNF125start31/stop129 in 50 mM Tris pH 7.0, 50 mM NaCl, 200 μM ZnCl2, 1 mM DTT, a set of standard triple resonance experiments for the sequential assignment was recorded using incremental non-uniform sampling50 (link) on Bruker 800 MHz Avance III HD spectrometer equipped with 5 mm TCI CryoProbe (Swedish NMR Centre) at 25 °C. For 13C15N-UbcH5a assignment in 50 mM Tris pH 7.0, 150 mM NaCl, 200 μM ZnCl2, 1 mM DTT, recording was performed with a Bruker 900 MHz Avance III HD spectrometer equipped with 5 mm TCI CryoProbe (Swedish NMR Centre) at 25 °C. Spectra were processed using MDDNMR51 (link) and the analysis was performed in CcpNmr Analysis.
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