Superose 6 increase 10 300 gl

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) (link). Glycerol was added to the sample at a final amount of 20% (v/v). Ponceau S at a final concentration of 0.001% (w/v) served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM tricine, 15 mM Bis-Tris/HCl, pH 7.0, 0.05% (w/v) sodium deoxycholate, 2 mM DTT, and 0.01% (w/v) dodecyl maltoside, whilst the anode buffer contained 50 mM Bis-Tris/HCl, PH 7.0, 2 mM DTT. An 8–15% linear polyacrylamide gradient gel was used, and electrophoresis was run under a N₂/CO₂ (90:10%) atmosphere with a constant 40 mA current (PowerPac^™ Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^™ Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^™ Unstained Protein Standard (Thermo Fischer Scientific, Driesch, Germany).
The determination of the oligomeric state by gel filtration was performed in triplicate on a Superose 6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.6, 2 mM DTT, 10% (v/v) glycerol at a flow rate of 0.4 ml/min, and in an anaerobic Coy tent containing an N₂/H₂ (97:3%) atmosphere. High molecular weight range gel filtration calibration kit (GE Healthcare, Munich, Germany) was used as the protein standard.

The hrCN PAGE protocol was adapted from Lemaire et al. (2018) [24 (link)] Glycerol was added to the sample at a final amount of 20% v/v. Ponceau S at a final concentration of 0.001% w/v served as a marker to follow the migration. The buffer composition for the electrophoresis cathode was the following: 50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7, 0.05% w/v sodium deoxycholate and 0.01% w/v dodecyl maltoside, while the anode buffer contained 50 mM Bis-Tris/HCl buffer pH 7. A 5 to 15% linear polyacrylamide gradient gel was used and electrophoresis was run with a constant 40 mA current (PowerPac^TM Basic Power Supply, Bio-Rad). After electrophoresis, protein bands were visualised with Ready Blue^TM Protein Gel stain (Sigma Aldrich, Hamburg, Germany). The native protein ladder used is NativeMark^TM Unstained Protein Standard (ThermoFischer Scientific, Dreieich, Germany).
Determination of the oligomeric state by gel filtration was performed on a Superose6 Increase 10/300 GL (GE Healthcare, Munich, Germany) in 25 mM Tris/HCl pH 7.4, 2 mM DTT, 10% glycerol at a 0.4 mL/min flow rate. High Molecular Weight range Gel Filtration Calibration Kit (GE Healthcare, Munich, Germany) was used as the protein standard.

The complex was on-column glutaraldehyde cross-linked28 (link) in 50 mM HEPES-NaOH pH 7.7 (4 °C), 200 mM NaCl, 1 mM EDTA as follows: 500 μl of a 0.25% glutaraldehyde solution was pre-injected onto a size-exclusion column (Superose 6 Increase 10/300 GL, GE Healthcare) and run for 20 min at a flow rate of 0.25 ml/min. The run was paused, the injection loop vigorously washed, UtpB injected, and the run was continued at the same flow rate. The on-column cross-linking procedure was optimized with UtpB purified using affinity tags on different subunits (YSK43 and YSK47, Supplementary Data 1). UtpB from both strains exposed to the pre-injected glutaraldehyde bolus exhibited the same elution behaviour as the non cross-linked complex (Supplementary Fig. 5a) and the protein complex eluted at 13.14 ml.

1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPc), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (PS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE) were obtained from Avanti Polar Lipids. His60 Ni Superflow Resins were purchased from Takara Bio USA and Superose 6 Increase 10/300 GL was from GE Healthcare. All other chemicals were acquired from Sigma.

For exchanging the detergent LMNG with amphipol, bd-II was supplemented with a threefold mass excess of amphipol A8-35 (Anatrace, Maumee, OH, USA). The sample was incubated at 4 °C for 2 h while stirring. Excess detergent was removed by an addition of a 40-fold mass excess BioBeads (SM-2 resin, BioRad, Feldkirchen, Germany) and further incubation of 3 h at 4 °C under stirring. Excess amphipols were removed by size exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare), equilibrated in 20 mM MOPS, 20 mM NaCl, pH 7.0. Peak fractions were pooled and used for further analysis. For cryo-EM the samples were supplemented with 1 μM aurachin D prior to placing them onto the cryo grids.