SEC-SAXS data collection was performed at RIKEN beamline BL45XU (15) in SPring-8 and at beamline SWING (16) in the French national synchrotron SOLEIL. At BL45XU in SPring-8, the purified protein was loaded onto a Superdex 200 Increase 3.2/300 column (GE Healthcare) in a 20-μl volume at 50 μl/min flow rate with a SEC buffer [40 mM tris-HCl (pH 8.0), 10% (w/v) glycerol, and 5 mM MgCl2] at 20°C. For the data collection of cyanomet FixL, 5 mM KCN was added to the SEC buffer, and the loading FixL sample was mixed with KCN at the final concentration of 5 mM. For the detection of the FixL-FixJ complex or the FixL-FixJN complex, SEC buffer containing saturating amounts of FixJ or FixJN (more than 40 μM, 10 times of the Kd value) was used to improve the affinity of FixL and FixJ. X-ray exposure was 1 s every 4 s with the incident beam energy 12.4 keV. Buffer frames (30 × 1 s) were averaged and subtracted from 30 × 1 s frames taken over the course of protein elution. The sample detector distance was 2 m, giving an angular momentum transfer range of qmin = 0.009 Å−1 to qmax = 0.5 Å−1. The flux density was about 2 × 1012 photons/s/mm. Scattering was collected on a PILATUS3 X 2M detector (Dectris). Data averaging and reduction were calculated by the program DataProcess installed in BL45XU. Measurements at SWING were performed as above but using an Agilent Bio SEC-3 4.6 × 300–mm column (Agilent) with 3-μm bead size and 300 Å pore size at 15°C after double purification on a Superdex 200 Increase 10/300 column (GE Healthcare) at 4°C. Beam energy was 12 keV, and sample detector distance was 1.8 m.

Rg and I0 calculations on the SEC-SAXS data were performed with AutoRg (43). Data of interest was averaged, and the Guinier estimation was performed in MATLAB and PRIMUS (fig. S10) (44). Distance distribution functions P(r) were calculated with GNOM (45) and ScÅtter based on agreement between real and reciprocal space Rg values (<3% difference) and fit to the experimental data. This was performed independent of crystallographic and homology model building. Bead models were generated with DAMMIN (46).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.