Superdex 200 increase 10 300 column

SEC-MALS analysis was performed with an HPLC system (Waters, Milford, MA, USA), coupled with UV- (Waters), Dawn8+ MALS- (Wyatt, Dernbach, Germany), and RI- (Shodex, Tokyo, Japan) detectors. Samples were filtered using 0.1 μm centrifugal filters (Millipore, Burlington, MA, USA) and injected onto Superdex 200 Increase 10/300 column (Cytiva, Marlborough, MA, USA), previously equilibrated with 20 mM Tris pH 7.5, 150 mM NaCl. Data were analyzed using Astra 7.0.

Snap-frozen purified sTMEM2 was thawed and rerun on a Superdex 200 increase 10/300 column (Cytiva). Peak fractions were concentrated to 9 mg/ml and a range of commercial crystallisation screens were set up using a Mosquito nanolitre liquid handler (STP Labtech). Large single crystals were obtained at room temperature using 0.1 M sodium acetate, pH 4.5, 30% polyethylene glycol 3000 as precipitant. The crystals were frozen in liquid nitrogen using precipitant solution supplemented with 20% ethylene glycol as cryoprotectant. Diffraction data were collected on beamline I04 at the Diamond Light Source (λ = 0.9795 Å) and processed using the
XIA2 DIALS pipeline (version 3.dev.661-g1a4ae04e6)
^{13 (link)}
. The structure was solved by molecular replacement using
PHENIX (version 1.18rc1_3769)
^{14 (link)}
and
AlphaFold 2.0 model
Q9UHN6 as search model
^{9 (link)}
. Manual rebuilding and refinement were done using
COOT (version 0.8.9.2)
^{15 (link)}
and PHENIX. A conservative refinement protocol using a single B-factor per residue gave the same
R_free as models with more parameters. Crystallographic data are summarised in
Table 1. Structural comparisons were done using the
DALI server
^{16 (link)}
. All software used in the study are freely available to academic users.

CSGALNACT2 ΔCC at a concentration of 4 mg/mL was injected onto a Superdex 200 Increase 10/300 column (Cytiva) connected to an Agilent 1260 Infinity system. The running buffer was 25 mM Na-HEPES pH 7.5, 150 mM NaCl and the flow rate was 0.5 mL/min. Light scattering and refractive index changes were monitored using in-line Wyatt Mini Dawn and Optilab T-rEX detectors (Wyatt Technology Corp). The data were analysed with the Wyatt ASTRA V software and gave an experimental mass of 109 kDa.

A protein sample (0.2 mg/ml) was injected in a Superdex 200 Increase 10/300 column (Cytiva) previously equilibrated in 20 mM Tris pH, 150 mM NaCl, 1 mM DTT, 5% Glycerol buffer (filtered through a 0.1 μm filter). The chromatographic eluent was monitored by three consecutive detectors in series: (1) a multi-wavelength UV-Vis absorbance detector Monitor UV-900 of the AKTA system (GE Healthcare) with a 10 mm path length flow cell, (2) a light scattering DAWN Heleos 8+ (Wyatt Technology) with detectors at eight different angles (from 32 to 141° from the source) using a linearly polarized GaAs laser operating at 665 nm and (3) an Optilab T-rEX (Wyatt Technology) differential refractive index detector with a laser wavelength of 658 nm. Data collection and analysis were performed using UNICORN 5.10 (GE Healthcare) and ASTRA 6.0.3 (Wyatt Technology) software packages.