Diffuse X-ray scattering: Offers protein clues

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  • Published: Oct 1, 2015
  • Author: David Bradley
  • Channels: X-ray Spectrometry
thumbnail image: Diffuse X-ray scattering: Offers protein clues


Diffuse X-ray scattering pattern. Credit:Van Benschoten et al.

Protein flexibility is essential for the functioning of enzyme, cellular signalling processes and protein-protein interactions. Now, diffuse X-ray scattering has been used to help report correlated atomic displacements in proteins.

Protein movements can range in scale from a few angstroms to many nanometres and encompass the kind of transitions that occur between side-chain rotamers, loop openings and closings, and rigid-body subunit rotations, any one of which may be the critical shift in shape that initiates or modulates a given biological process. Protein crystallographers almost routinely compare crystal structures to spot the differences between conformations and the before and after shapes of proteins and endless hypotheses are put forward to explain how a given movement correlates with a particular protein function.


However, often, only a single form is available from the crystal structure and any evidence for concerted motion can only be inferred from the electron density data. Thankfully, diffuse X-ray scattering can assist in the reporting of correlated atomic displacements, although new approaches and computational techniques are needed to glean the most information from the data. Indeed, recent advances have boosted the potential for accurately measuring diffuse scattering, but computational modelling and validation tools are still needed to quantify the agreement between experimental data and the parameterization of crystalline disorder.

Now, Andrew Van Benschoten and James Fraser of the University of California San Francisco and colleagues in Australia, France and elsewhere in the USA, Pavel Afonine, Thomas Terwilliger, Michael Wall, Colin Jackson, Nicholas Sauter, Paul Adams and Alexandre Urzhumtsev believe they have the right tool for the job in the form of "phenix.diffuse", which they suggest addresses this need by calculating diffuse scattering from Protein Data Bank (PDB)-formatted structural ensembles. They report details in the journal Acta Crystallographica Section D (Biological Crystallography).

Blooming structures

Given the advent of powerful modelling tools, it is the lack of high-quality three-dimensional X-ray data that has become the main bottleneck in the flow of diffuse scattering analysis. One of the main problems in data collection is that the long X-ray exposure times needed to reveal diffuse features cause a distortion, known as blooming, around any saturated Bragg spots in diffraction images collected with commercially available charge-coupled device (CCD) area detectors. Blooming interferes with accurate diffuse intensity measurements but it can be mitigated either by reconfiguring some of the charge collection elements in each pixel as a drain to channel away excess charge (although this is not an option available at the moment in commercial CCDs). Alternatively, it is possible to carry out a series of shorter exposures rather than one single long shot. Each of those solutions comes with its own set of problems. A better approach would be to use pixel-array detectors to obtain more accurate measurements of the diffuse signal as they have a higher dynamic range as well as very small point-spread functions. But, these devices are not as common as the conventional CCD type.

The "phenix.diffuse" system allows the team to process diffuse scattering data from raw image frames to complete a reciprocal-space map. The researchers point out that instead of being included in the background correction in estimated Bragg peak intensities, the diffuse intensities will increase the data available for refinement, enable more accurate quantification of interatomic distances and allow the simultaneous refinement of multiple coupled protein motions. They have demonstrated proof of principle by successfully modelling the enzyme glycerophosphodiesterase, GpdQ, and obtained new insights into the molecular mechanism and allostery of this protein. "These methods demonstrate how, in principle, X-ray diffuse scattering could extend macromolecular structural refinement, validation and analysis," the team concludes.

Related Links

Acta Cryst D 2015, 71, 1657-1667: "Predicting X-ray diffuse scattering from translation-libration-screw structural ensembles"

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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