Diffract and destroy: Fluctuation X-ray scattering

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  • Published: Jun 1, 2013
  • Author: David Bradley
  • Channels: X-ray Spectrometry
thumbnail image: Diffract and destroy: Fluctuation X-ray scattering

Shine a light

Fluctuation x-ray scattering is the basis of a new technique for rapidly modeling the shapes of large biological models, here demonstrated (gray envelopes) using existing diffraction data superposed on known high-resolution structures. Top left, lysine-arginine-ornithine (LAO) binding protein; top right, lysozome; bottom left, peroxiredoxin; and, bottom right, Satellite Tobacco Mosaic Virus (STMV). (Credit: Image courtesy of DOE/Lawrence Berkeley National Laboratory)

Fluctuation X-ray scattering could help fill the gaps in our knowledge of protein structure thanks to the short bursts of radiation possible with free electron laser sources, allowing data to be obtained from non-crystallisable proteins in their native, fluid state albeit with the sacrifice of the protein itself.

Function is mostly about form when it comes to proteins and form is structure, understanding how structure change can then lead to a better understanding of how a specific protein works. Our best tools for the job of determining protein structure rely on our being able to make crystals of the biomolecule and for more than 80,000 proteins this has worked well over many decades of scientific research. , as molecules in the native state do their jobs. Unfortunately, there are about two million proteins in the human body that cannot be crystallized and for the majority of them, even a low-resolution structure has not yet been determined.

Collaborative light

Writing in the journals Foundations of Crystallography and Physical Review Letters, Peter Zwart and his colleagues at the US Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab), working with collaborators from Arizona State University, the University of Wisconsin-Milwaukee, and DOE's Pacific Northwest National Laboratory (PNNL) promise a way to obtain structural information from proteins even in their native, more fluid, state.

The technique of "diffract before destroy" has been useful in looking at biological macromolecules using free-electron lasers (FELs) such as the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory. The intense X ray pulses from these devices last quadrillionths of a second, but such a pulse is enough to freeze the action as a protein as it rotates in the fluid state, for instance before it is annihilated by the energy. It is not crystallisation, but it is close, the pulses are shorter than the rotation time and so can essentially lock on to a molecular structure and provide useful experimental data as a proxy for full-blown X-ray crystallography.

Diffract then destroy

"It's a technique called 'diffract before destroy,' because the data is collected before the particle literally blows apart," explains Zwart, who is the lead scientist for the Berkeley Center for Structural Biology at the Advanced Light Source. "FELs have shown they can derive structures from single particles, each hit with a single pulse, but there are major challenges to this approach."

However, Zwart and his colleagues were not satisfied with snapping single particles, they wanted to get information from many particles to feed the data to computer program that would combine the myriad diffraction patterns and offer them detailed insights into the structures adopted by the molecules in solution. Their approach, fluctuation X-ray scattering (fXS), has now shown how data obtained this way with FELs can yield low-resolution structures of various biomolecules as they exist close to their natural state. This offers much greater confidence levels than is currently possible with less powerful synchrotron light sources.

"Our algorithm starts with a trial model and modifies it by randomly adding or subtracting volume until the shape of the model achieves the optimum fit with the data," Zwart explain. The team has effectively tested this trial-and-error optimization technique on known configurations at the LCLS and demonstrated that it can resolve the shapes of individual macromolecules with fXS data alone. The researchers have also applied their approach to mixtures, which allows them to disentangle clues regarding individual components in large biological systems. Zwart and his colleagues hope that fluctuation X-ray scattering will become an indispensable tool for determining how mixtures of different proteins behave independently or together.

"The next step in the research is to get experimental data and show that we can do this on large macromolecular assemblies, such as virusses and virus like particles," Zwart told SpectroscopyNOW.

Related Links

Acta Cryst Sect A Found Crystallogr 2013, 69, online: "Three-dimensional single-particle imaging using angular correlations from X-ray laser data"

Phys Rev Lett 2013, 110, 195501: "Component Particle Structure in Heterogeneous Disordered Ensembles Extracted from High-Throughput Fluctuation X-Ray Scattering"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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