Protein Envelope Determination

3D envelopes were established using an automated sequence of measurements, including three different proteins with low volume and dilute solutions.

Introduction

Small-angle X-ray scattering is complementary to other structural biology methods. It supplies good information about the shape of proteins in solution and associated parameters, including macromolecule interactions and dynamics. The high throughput and reliability of the measurements are vital requirements for the ability to run samples in a structural biology laboratory.

This article shows the quality of three-dimensional envelopes gathered on three protein samples (bovine serum albumin (BSA), lysozyme, and thyroglobulin) using an automated measurement sequence, measured on the BioXolver.

Measurements & Results

Concentration series, employing the in-line pipetting robot, which permits automatic sample loading and cell cleaning in a cycle time, which is less than two minutes, three concentrations per sample ran successively. For the measurements, 5 µL per sample condition were utilized.

To define sample quality, data treatment through the automated software package based on ATSAS1 enables the fast calculations of the corresponding 3D envelopes for each concentration.

The full sequence of measurements was completed in under 90 minutes, including the three concentrations per buffer and sample measurements, from the loading of the well plate through to the generation of the final 3D envelopes. To emphasize data quality, additional data processing was performed using ATSAS.

The 3D protein envelopes produced by DAMMIN2 for the lowest measured concentrations of BSA and lysozyme can be seen in Figures 1 and 2. By overlaying the homologous crystal structure for both samples comparison of the calculated shape to existing crystallographic data is supplied. Juxtaposition shows good consistency of the estimated shape.

This shows the ability to establish 3D envelopes from weakly scattering samples in short measurement time (five minute exposure time). Comparison between the synchrotron SAXS data and BioXolver measurements for thyroglobulin sample can be observed in Figure 3.

The same analysis procedure, which generates DAMMIF/DAMMIN models, was applied to both SAXS data sets. The final 3D envelopes are extremely similar and exhibit the good quality of the results gathered on the BioXolver in a five minute exposure time.

Conclusion

The capability of the BioXolver to produce high-quality results reliably, with high throughput and employing small volumes of protein solution is seen in the results. It paves the way for routine characterization of biological macromolecules in the laboratory, supplying quick feedback for formulation studies and other research.

3D envelope from a lysozyme solution (Mw = 14.3 kDa).

Figure 1. 3D envelope from a lysozyme solution (Mw = 14.3 kDa).

3D envelope from a BSA solution (Mw = 66 kDa).

Figure 2. 3D envelope from a BSA solution (Mw = 66 kDa).

Comparison of 3D envelopes of thyroglobulin (Mw = 669 kDa).

Figure 3. Comparison of 3D envelopes of thyroglobulin (Mw = 669 kDa).

References and Further Reading

  1. D. Franke et al. (2017) J. Appl. Cryst. 50
  2. D. I. Svergun (1999), Biophys J. 2879-2886.
  3. www.rcsb.org
  4. www.sasbdb.org

About Xenocs

Xenocs is a supplier of x-ray scattering equipments (SAXS/WAXS) for characterizing the nanostructure and morphology of materials at the nanoscale. Such equipments are used for research, development, and production of advanced materials in various domains such as nanoparticles & colloïds, polymer, food science, cosmetics or biostructural research.

Created in 2000 as a spin off from Institute Laue Langevin, the company started activity offering its customers innovative X-ray sources, optics and collimation solutions for X-ray characterization of nanomaterials and nanostructures.

Xenocs delivered its first x-ray scattering equipment in 2008, setting new standards for the possibilities of using SAXS in the laboratory for characterization at the nanoscale.

Working closely with both academic and corporate customers, Xenocs has continuously focused on providing value through performance and ease of use, while also creating a global sales and service organization.

In 2016, Xenocs bought Saxslab with operations in Denmark and in Massachusetts, making it the leading provider of SAXS equipments worldwide. Xenocs headquarters are located in France, and the company has subsidiaries in USA, Denmark and Singapore as well as a strong network of local contacts.


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Last updated: Aug 3, 2020 at 7:18 AM

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