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Synchrotron SAXS
data from solutions of
Human Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 protein DH/PH domain and DEP1 module (P-Rex2 DH/PH-DEP1)
in
20 mM HEPES, 200 mM NaCl, 2 mM DTT, 1% glycerol, pH 7
were collected
on the
12.3.1 (SIBYLS) beam line
at the Advanced Light Source (ALS) storage ring
(Berkeley, CA, USA)
using a Pilatus3 X 2M detector
at a sample-detector distance of 2.1 m and
at a wavelength of λ = 0.1127 nm
(I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle).
In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A sample
at 2 mg/ml was injected at a 0.65 ml/min flow rate
onto a Shodex KW-800 series column
.
750 successive
2 second frames were collected.
The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.
Small-angle X-ray scattering (SAXS) experiments were performed at the SIBYLS beamline 12.3.1 at the Advanced Light Source. This beamline has additional inline instrumentation and detectors coupled to a size exclusion column. Purified P-Rex2 DH/PH-DEP1 was concentrated to 2 mg/ml and then buffer exchanged into 20mM HEPES (pH 7), 200mM NaCl, 2mM DTT, and 1% glycerol SEC running buffer. The X-ray wavelength was set to 1.127 Å and the sample-to-detector distance to 2,100 mm, which gives scattering vectors (q) ranging from 0.01 Å-1 to 0.4 Å-1. The scattering vector is q = 4πsinθ/λ, where 2θ is the scattering angle. The SAXS flow cell was coupled to an Agilent 1260 Infinity HPLC system using a Shodex PROTEIN KW-802.5 SEC column equilibrated with the running buffer with a flow rate of 0.65 ml/min. For SAXS measurements, 2 sec X-ray exposures were collected continuously during a 25 min elution. All frames for analyses had one SAXS frame corresponding to the running buffer before the detection of a peak subtracted from each.
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s, nm-1
