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Analytical Chemistry

Improving Protein Crystallography Using Smudges In X-Ray Diffraction Data

Spectroscopy: New technique takes advantage of long-unused data

by Jyllian Kemsley
February 10, 2016 | A version of this story appeared in Volume 94, Issue 7

Examples of diffraction data produced by perfectly ordered and disordered crystals.
Credit: Nature
In a perfectly ordered crystal (molecules represented by ducks), X-ray diffraction produces bright Bragg peaks (top). When the crystal has disorder, such as when molecules shift translationally, continuous diffraction appears (bottom).

Vague, rippling patterns in X-ray diffraction data long considered useless for producing high-resolution protein structures instead can help improve them, according to a new study (Nature 2016, DOI: 10.1038/nature16949).

Using these data patterns not only could increase the resolution of structures, but also may allow crystallographers to solve protein structures without using heavy-atom derivatives or the structure of a closely related protein as a starting point, two common methods employed to analyze proteins.

“This paper will send swathes of macromolecular crystallographers rummaging through volumes of discarded diffraction data looking for the ‘smudgy’ speckle signature in the hopes of using this new diffractive imaging approach to salvage long-abandoned projects,” comments Martin Caffrey, a biochemistry professor at Trinity College Dublin.

When X-rays interact with a crystal, they scatter off the electrons in the molecules and form a pattern of bright spots known as Bragg peaks. Crystallographers use the peaks to determine the atomic structure of the molecules.

But crystals, especially those of proteins, often aren’t perfectly ordered. This leads to continuous diffraction—rippling patterns generated by the incoherent sum of X-rays scattered from individual molecules or molecular complexes.

A team led by Henry N. Chapman of the Center for Free-Electron Laser Science at the German Electron Synchrotron has now demonstrated a method to extract structural data from continuous diffraction, solving the structure of photosystem II to a resolution of 3.5 Å. Analysis using only the Bragg peaks of the same data yields a structure with a lower resolution of 4.5 Å.

The approach works when disorder in crystals comes from translational and some rotational displacement of molecules. But it still doesn’t help when the disorder is caused by dynamic motion or variable composition, so proteins that are more flexible than photosystem II may not be as amenable to the analysis.

Chapman and colleagues collected the photosystem II data from a stream of micrometer-sized crystals using femtosecond X-ray pulses generated by the free-electron laser at the U.S. SLAC National Accelerator Laboratory. It remains to be seen whether the method works as well for conventional experiments that use larger crystals with longer X-ray exposure. “You would need to minimize the background noise from other things in the beam,” such as the crystal holder, Chapman says. “I think that’s probably the main reason why continuous diffraction hasn’t really been studied or used much before—it’s been this very shadowy thing that was hard to pin down.”



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