At present, the small-angle X-ray scattering (SAXS) technique is widely used for the analysis of biological systems, especially proteins in solution. The macromolecular shape conformation of proteins can be determined with the help of SAXS measurements through envelope reconstruction.
SAXS/WAXS examination of the protein Sub-unit F of the eukaryotic V1V0 ATPase was carried out using the Xeuss 2.0 SAXS/WAXS system. This instrument can capture extremely high quality data, enabling accurate resolution of the protein structure.
Measurements and Results
Measurements were performed on 2, 5, 10 and 17 mg/ml concentration solutions, by means of the buffer 50 mM HEPES (pH 7.0), 300 mM NaCI and 1 mM DTT, on the Xeuss 2.0 SAXS/WAXS system in combination with a photon counting Pilatus 1M pixel detector.
The evolution of I (q=0) as a function of the concentration is depicted in figure 1. An obvious inconsistency in the scaling away of the intensity from the linear behavior may indicate one or both of these- the presence of aggregates for concentrations more than 10 mg/ml or the impact of inter-particle interactions. The 10 mg/ml concentration was chosen as it offers higher scattering without additional contribution to that of the protein.
Figure 1. Evolution of I(q=0) as a function of concentration. The black line is a guide to the eye, passing through 0,0.
Figure 2 displays the resulting 1D curves. Figure 3 shows the pair-distance distribution function p(r), determined using the GNOM
1 software. The curve shows a principal maximum at 2.8 nm and a protruding shoulder from 5.5 nm up to 7.0 nm. These results are consistent with synchrotron measurements 2.
Figure 2. 1D curve from Subunit F, c = 10 mg/ml. Exposure time = 6 x 600s. Logarithmic rebin of data.
Figure 3. Pair-distribution function.
The low-resolution structure of sub-unit F is shown in Figure 4 restored
ab-initio with the help of DAMMIN 1, with just one iteration. The protein can be seen as a two-domain molecule having a large egg-like-shape, linked through a long stalk to a tiny hook-like region at the end. This is almost identical to the acquired external envelope reconstruction from synchrotron data 2.
Figure 4. Structure ab-initio modeling using DAMMIN.
Comparison between the data generated using the Xeuss 2.0 and the data obtained from the SAXS beamline X33 at the DORIS III storage ring at DESY4 was drawn by Dr. D. Svergun, with the same experimental conditions such as concentration and buffer. It was reported by Dr. D.Svergun that the Xeuss data matched up in a consistent manner with the synchrotron results.
Petoukhov et al. J. Appl. Cryst., 2007, 40 s223-s228.
S. Basak et al., Biochimica et Biophysica Acta 1808 (2011) 360-368.
Deutsches Elektronen-Synchrotron, EMBL, Hamburg.
This information has been sourced, reviewed and adapted from materials provided by Xenocs.
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