Sparky

NMR spectra were acquired on a Bruker Avance 600 MHz spectrometer with a triple-resonance cryoprobe at 25°C. To assist the assignment of the backbone and side chain NH signals in each mutant, ¹⁵N-labeled samples were prepared in 45 mM sodium phosphate (pH 7) with 100 mM final ionic strength in 92% H₂O/8% ²H₂O. Natural abundance samples of the mutants were prepared in the same buffer to assist the assignment of the heme substituent signals. OmcF wild-type samples were also prepared in the same buffer (pH 6.1 and 9.4) in ²H₂O (99.9%) to study the pH dependence of the heme substituents’ signals.
Reduction of the proteins was achieved by adding an equimolar solution of sodium dithionite, after degassing the samples with a continuous flow of argon. The full reduction of the samples was confirmed by 1D ¹H NMR. 2D ¹H,¹⁵N-HSQC spectra were acquired for ¹⁵N-labeled samples, whereas 2D ¹H, ¹H-TOCSY (60 ms) and 2D ¹H, ¹H-NOESY (80 ms) were acquired for natural abundance samples.
The water signal was used to calibrate the ¹H chemical shifts. ¹⁵N chemical shifts were calibrated using indirect referencing (Wishart et al., 1995 (link)). The data were processed using TOPSPIN (Bruker Biospin, Karlsruhe, Germany) and analyzed with Sparky (TD Goddard and DG Kneller, Sparky 3, University of California, San Francisco, CA, United States).

Spectra were recorded on Bruker 600, 700 and 850 MHz NMR spectrometers equipped with CyroProbes. ¹H spectra were acquired with 128–1024 scans and a relaxation delay 2 s. TOCSY (20 ) and DQF-COSY (21 ,22 (link)) spectra were collected in D₂O at 288 K. NOESY spectra with H₂O suppression [Watergate W5 with gradients (23 (link),24 )] were collected in D₂O at 288 K and 90%/10% H₂O/D₂O at 288 and 298 K, respectively, with mixing time of 100, 120 and 300 ms. These spectra comprised 2048 × 512 complex points and were acquired with a 2 s relaxation delay. JR-HMBC spectra (25 (link)) were acquired at 298 K with 8 K scans and comprised 2048 × 48 complex points. ¹H chemical shifts were referenced to 2,2-dimethylsilapentane-5-sulfonic acid at 0 ppm.
³¹P NMR were collected at 288, 298 and 310 K and referenced to external 85% H₃PO₄. ³¹P–¹H COSY (26 (link)) and HSQC (27 (link)) spectra were recorded at 298 and 310 K, respectively. ³¹P assignments were accomplished using a combination of ¹H–¹H NOESY, COSY, TOCSY and hetero-nuclear ³¹P–¹H COSY data. Spectra were processed using Bruker TopSpin 3.2 and analyzed using SPARKY (28 ).

Membranes were washed twice with 10 mM phosphate buffer pH 6.8 (in H₂0) to remove any Tris from the lysis buffer and collected by centrifugation at 125,000 xg for 1 – 2 hrs before packing into a 1.3 mm rotor for magic angle spinning (MAS). YidC samples were measured on a 800 MHz wide-bore or 700 MHz narrow-bore spectrometer (Bruker Biospin, Germany) with a 1.3 mm ¹H, ¹³C, ¹⁵N MAS probe at 55 kHz MAS frequency, and with a set temperature of 253 K (corresponding to an effective temperature of ∼ 30 °C). Spectra were referenced against adamantane (Harris et al., 2008 ) and histidine (Wei et al., 1999 ) powders. Data were processed with TopSpin 3.0 (Bruker Biospin) and analyzed using Sparky (Goddard and Kneller, 2008 ). Chemical shift predictions were made with Shiftx2 (Han et al., 2011 (link)) and FANDAS (Gradmann et al., 2012 (link)) using atomic model PDB ID 3wvf (Kumazaki et al., 2014 (link)).

For NMR sample preparation, MIER1(aa:1–177) was uniformly labelled by overexpression in M9 minimum medium, containing ¹⁵NH₄Cl as the sole nitrogen source. The expression and purification of labelled MIER1 was performed as described above. MIER1(aa:1–177) (200 μM) with 10% D₂O in a final volume of 330 μl of PBS buffer was placed in 3 mm NMR tubes (Norell). (¹⁵N–¹H)-HSQC spectra were measured on a Bruker AVIII-600 MHz spectrometer equipped with a cryoprobe. Data were processed using Topspin (Bruker) and analysed with Sparky. H2A:H2B dimer was added to the labelled MIER1(aa:1–177) protein at the ratio of 1:1 and incubated for 30 min at 4°C. The MIER1(aa:1–177):H2A:H2B complex was then purified on a Superdex S200 column before collecting (¹⁵N–¹H) HSQC NMR data.

All experiments were performed in a Bruker Ascend 850 MHz magnet at the University of British Columbia. Data were processed and analyzed using the programs TopSpin (BRUKER Ltd.) and Sparky (Goddard and Kneller ) respectively. Proton correlation (COSY), total correlation (TOCSY), nuclear Overhauser effect (NOESY) spectroscopy as well as proton-carbon heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) spectroscopy were performed on synthetic, 5 mM DSSHAFTLDELR peptide in 25 mM sodium phosphate buffer (pH 6.5) with 5% D₂O at 10, 20, 30, and 37°C. Spectral assignment was performed manually and chemical shift data were assessed by MICS (Motif Identification from Chemical Shifts) software (Shen and Bax, 2012 (link)).

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).

1D and 2D NMR data were collected on a Bruker AV-600 spectrometer (QCI cryoprobe). The w5 water suppression was used. 2D NOESY were collected at temperatures of 20, 25, and 35 °C with mixing times of 80, 150, 300, and 350 ms in 90% H₂O/10% D₂O solutions. Moreover, some representative NOE build-up curves have been determined (Supplementary Fig. 42). The NOE distance calibration was done using the cytosine H5/H6 distance (2.4 Å). DQF-COSY spectrum was collected at 25 °C. ¹H-¹³C HSQC spectra were collected using the hsqcetgpsi pulse sequence with ¹J_{(C, H)} = 145 Hz. Chemical shift calibration was done indirectly for ¹³C relative to DSS and directly for ¹H according to the water signal relative to DSS. The NMR data were processed and analyzed with Topspin 4.1.1 (Bruker) and Sparky (UCSF), respectively. For 2D NOESY spectra, 480–580 increments were accumulated in the indirect (F1) dimension and 2048 points in the direct (F2) dimension. The spectral widths were 16.5 ppm and 10 ppm for F2 and F1 dimensions, respectively, or 16.5 ppm for both dimensions. The processing parameters were 1024 points for F1 and 2048 points for F2, with a Sine bell shift (SSB) of 3 in both dimensions and the QSINE window function.