Protein Structure Determination with Small Angle X-Ray Scattering

Topics Covered

Introduction
Measurements and Results
Conclusions
References
About Xenocs

Introduction

The small-angle X-ray scattering (SAXS) technique is now quite popular for the analysis of biological systems, particularly proteins in solution. SAXS measurements can help to determine the macromolecular shape conformation of proteins, via envelope reconstruction.

SAXS/WAXS investigation of the protein Sub-unit F of the eukaryotic V1V0 ATPase was performed using the Xeuss 2.0 SAXS/WAXS system. This instrument is capable of capturing extremely high quality data, allowing accurate resolution of the protein structure.

Measurements and Results

Measurements were done on 2, 5, 10 and 17mg/ml concentration solutions, using the buffer 50mM HEPES (pH 7.0), 300mM NaCI and 1mM DTT, on the Xeuss 2.0 SAXS/WAXS system using a photon counting Pilatus 1M pixel detector.

Figure 1 shows the evolution of I (q=0) as function of the concentration. A clear inconsistency in the scaling away of the intensity from the linear behaviour may show either the impact of inter-particle interactions or the presence of aggregates for concentrations more than 10mg/ml. The 10mg/ml concentration was selected for offering higher scattering without additional contribution to that of the protein.

Figure 1. Evolution of I(q=0) as function of concentration. The black line is a guide to eye, passing through 0,0.

Figure 2 shows the resulting 1D curves. Figure 3 shows the pair-distance distribution function p(r), determined using the GNOM1 software. The curve shows a principal maximum at 2.8nm and a protruding shoulder from 5.5nm up to 7.0nm. These results are consistent with synchrotron measurements2.

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 seen in Figure 4 restored ab-initio with the help of DAMMIN1, with a single 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 very similar to the obtained external envelope reconstruction from synchrotron data2.

Figure 4. Structure ab-initio modeling using DAMMIN

Conclusions

Data generated using the Xeuss 2.0 was compared by Dr D. Svergun with data obtained from the SAXS beamline X33 at the DORIS III storage ring at DESY4, with the same experimental conditions such as concentration and buffer. Dr D.Svergun reported that the Xeuss data matched up consistently with the synchrotron results.

References

  1. Petoukhov et al. J. Appl. Cryst., 2007, 40 s223-s228.
  2. S. Basak et al., Biochimica et Biophysica Acta 1808 (2011) 360-368.
  3. Deutsches Elektronen-Synchrotron, EMBL, Hamburg.

About Xenocs

Since its creation in 2000, Xenocs has been dedicated to offering its customers innovative solutions for X-ray characterization of nanomaterials and nanostructures.

Xenocs has over 14 years' experience in the development and production of high performance instruments for characterizing the nanostructure and morphology of materials.

Its new generation of SAXS system, the  Xeuss 2.0, combines small and wide-angle X-ray scattering techniques (SAXS/WAXS) in a proprietary technology which is helping the company lead the way to the ultimate laboratory nanostructure characterization tool.

The company also launched this year a new vertical SAXS/WAXS system called the Nano-inXider which comes in a compact package for integration in any lab environment.

Xenocs is operating worldwide through a strong network of distributors ensuring local support and close contact to customers. Hundreds of research groups around the world as well as many renowned Original Equipment Manufacturers of the X-ray analytical market trust Xenocs to provide them with the best solution to move ahead in their research activities and industrial applications.

Xenocs is a spin-off company from the Laue Langevin Institut in Grenoble, France.

This information has been sourced, reviewed and adapted from materials provided by Xenocs.

For more information on this source, please visit Xenocs.

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