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Diffractive Imaging with Imperfect Crystals

Scatterings image

Slightly disordered crystals of photosystem II produce a complex continuous diffraction pattern (right) under X-ray light that contains more data than the Bragg peaks alone (left). Credit: Eberhard Reiman / DESY

Researchers from Germany, Greece and the United States have found a way to use the “noise” from X-ray crystallography of imperfect crystals to produce sharp, 3-D images of large protein complexes without the usual need for molecular phase data (Nature, doi: 10.1038/nature16949). The scientists say that this new technique, which combines methods for X-ray diffraction of crystals and X-ray imaging of single molecules, could be used to better understand the large cell-membrane proteins involved with metabolic processes, like photosynthesis and catalysis, to create cleaner energy sources and improve drug development.

The large-biomolecule dilemma

X-ray crystallography requires crystallizing many copies of the same molecule in the same phase (e.g., left- versus right-hand orientation). When X-rays are directed at the crystal, they scatter and form a diffraction pattern with bright spots called Bragg peaks. Researchers use these peaks to determine the molecule’s 3-D atomic structure.

X-ray crystallography has been used to identify the structure of tens of thousands of molecules, but it is not ideal for imaging large biomolecules because they don’t naturally form perfect crystals (imperfections cause continuous diffraction “noise” and reduce the number of Bragg peaks in the scatter pattern), and because it’s nearly impossible to determine their phase (information about the molecule’s orientation is needed to transpose Bragg peaks into a 3-D atomic-level image).

Getting signal from the noise

Led by DESY scientist Henry Chapman from the Center for Free-Electron Laser Science (Germany), the researchers found that the continuous diffraction patterns caused by the displacement of molecules in a crystal lattice made from large photosystem II proteins may actually be carrying information about the large biomolecule’s phase that could increase image resolution. Although displaced, the molecules would still have the same orientation within the crystal, so the continuous diffraction pattern from the scattered X-rays is actually the diffraction pattern of an individual molecule in a specific phase, amplified many times over.

When they combined Bragg peak data with continuous diffraction data, the scientists were able to produce an image of photosystem II with a spatial resolution of 3.5 Å, an improvement over the 4.5 Å using only the Bragg peak data. In their paper, the researchers say that their work shows that continuous diffraction “can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography.”

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