Avance neo 700 mhz

All NMR data were collected at 298 K on a Bruker Avance Neo 700 MHz and a Bruker Avance III 850 MHz spectrometers equipped with triple-resonance cryogenic probes. Sequential backbone assignments were obtained using Transverse relaxation-optimized spectroscopy (TROSY) versions (Pervushin et al. 1997 (link)) of conventional three-dimensional experiments (HNCO, HN(CA)CO, HNCA, HN(CO)CA, HN(CO)CACB, HNCACB and HN(CA)CB) (Sattler et al. 1999 (link)). IVL methyl group assignments were obtained using a two-dimensional constant time (CT) ¹H-¹³C_methyl HMQC (Tugarinov and Kay 2003 (link)) and three-dimensional HMCMC, HMCM(C)CB, HMCM(CC)CA and HMCM(CCC)CO (Sinha et al. 2013 (link)). Additionally, a 3D 13Ch-13CH3 SOFAST-HMQC-NOESY-HMQC with a mixing time of 200 ms (Rossi et al. 2016 (link)), a 3D 15N-resolved NOESY with a mixing time of 400 ms, and a 3D 13C-resolved NOESY with a mixing time of 400 ms were used. All methyl assignment experiments were recorded with non-uniform sampling (NUS). NMR data were processed with Topspin 3.2 (Bruker), or with NMRPipe (Delaglio et al. 1995 (link)) and SMILE (Ying et al. 2017 (link)) and analyzed with Sparky (Lee et al. 2015 (link)).

Amide H/D exchange rates in both ¹⁵N labelled apo-WBSCR27 and complex WBSCR27-SAH were measured using heteronuclear ¹⁵N–¹H NMR spectroscopy (at 35°C and pH 7.0) on a Bruker AVANCE Neo 700 MHz spectrometer. The details of the H/D exchange rate measurements and the calculation of the protection factors of the amide H_N atoms are given in the Supplementary Data.

NMR spectra were acquired at 298K on a Bruker Avance NEO 700 MHz spectrometer equipped with a Z-gradient cryoprobe. The spectra were processed with the Bruker TOPSPIN 4.0.5 software packages. For NMR studies of compound 2a with VPS29-VPS35, 5 μl from a DMSO-d₆ stock 20 mM solution were dissolved in 175 μl of buffer (50 mM KH₂PO₄, 50 mM Na₂HPO₄, pH 8.0, 150 mM NaCl, 2 mM DTT) and 20 μL of ²H₂O. Saturation Transfer Difference spectra (¹H spectral window = 16 ppm; relaxation delay = 3.0 s; number of points = 32K) were acquired with 1024 scans with on-resonance irradiation at −1.0 ppm for selective saturation of protein resonances, and off-resonance irradiation at 40 ppm for reference spectra. A train of 40 Gaussian shaped pulses of 50 ms with 1 ms delay between pulses were used, for a total saturation time of 2 s. 1D ¹H spectra were recorded using a 1 s presaturation pulse with a B₁ power of 200 Hz. WaterLOGSY experiments (WL) were acquired using the ePHOGSY sequence^{33 (link)}. WL experiments (¹H spectral window = 16 ppm; relaxation delay = 3.0 s; number of points = 16K, scan = 1024) employed a 20 ms selective Gaussian 180° pulse at the water signal frequency and a NOE mixing time of 1 s. Both for STD and WaterLOGSY, FIDs were multiplied by an exponential weighting (lb = 5 Hz) before Fourier transformation.