Lycksell M,
Rovšnik U,
Bergh C,
Johansen N,
Martel A,
Porcar L,
Arleth L,
Howard R,
Lindahl E,
Proceedings of the National Academy of Sciences
118(37):e2108006118
(2021)
DOI
SASDL53 – Gloeobacter violaceus Ligand-Gated Ion Channel (GLIC) at pH 7.5 measured with continuous-flow SEC-SANS
SANS data from solutions of Gloeobacter violaceus ligand-gated ion Channel (GLIC) at pH 7.5 measured with continuous-flow SEC-SANS in D2O, 20 mM Tris, 150 mM NaCl, and 0.5 mM matched-out deuterated DDM were collected on the D22 small-angle neutron scattering diffractometer at the Institut Laue-Langevin (ILL; Grenoble, France) using a Reuter-Stokes 3He detector at a wavelength of λ = 0.6 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). The SEC parameters were as follows: A 300.00 μl sample at 4 mg/ml was injected onto a GE Superdex 200 Increase 10/300 column at 10°C. 44 successive 30 second frames were collected through the SEC elution peak. The ion channel GLIC was expressed in Escherichia coli and purified using H2O based buffers and n-Dodecyl β-D-maltoside (DDM) detergent. The protein was exchanged to the D2O based buffer with deuterated DDM (d-DDM) in the SEC-SANS gel filtration. The d-DDM has been deuterated to have the same scattering length density as D2O (matched-out deuteration). The gel filtration was run with a 0.3 ml/min flow speed to allow for detergent exchange prior to the SANS measurement. Upon peak detection by UV-vis absorbance at 280 nm the flow was slowed to 0.05 ml/min for the duration of the peak, after which the flow was increased to pass the remainder of the column volume. Two SEC-SANS runs were performed, one with a detector distance of 2.8 m, and the other with a detector distance of 11.2 m. Data reduction and buffer subtraction were performed using GRASP version 9.04, utilizing collected frames preceding the protein peak for the buffer background. Data were corrected for the empty cuvette and background, and scaled by their transmission and thickness. They were scaled to absolute intensity by direct flux measurement. The intensities were scaled to account for differences in protein concentration between the detector distances. In merging the data from the two detector distances, the data from 11.2 m was used up to 0.7 1/nm, above which the data from 2 m was used. A small additional constant was subtracted as a final adjustment to the background. Guinier analysis and molecular weight estimation were performed in Primus, selecting the Guinier range with Autorg and the Bayesian inference molecular weight. The pair-distance distribution was calculated using BayesApp (https://somo.chem.utk.edu/bayesapp/), set to fit the background and with no specified maximum diameter.
s, nm-1
