In keeping with the adage, “If life gives you lemons, make lemonade,” an international team of scientists has shown that if X-crystallography relies on low-quality crystals, it can still derive high-quality structural information. In fact, resolutions can be achieved that surpass the Bragg diffraction limit.
The key, it turns out, is to make the most out of continuous diffraction data, which is ordinarily considered a nuisance in crystallographic analysis. Continuous diffraction data could be obtained from a single molecule, but would be too weak to yield any kind of analysis. But if such data could be combined from a collection of molecules, analyses would be possible. Each of the molecules in the collection, however, would have to be misaligned only in the translational sense. That is, the molecules could not be misaligned rotationally or differ intramolecularly.
With these limitations in mind, scientists based at the Center for Free-Electron Laser Science, DESY, in Germany “read” the atomic structure of complex biomolecules by crystallography without the usual need for prior knowledge and chemical insight. “This discovery has the potential to become a true revolution for the crystallography of complex matter,” said the chairman of DESY's board of directors, Professor Helmut Dosch.
The work of the DESY-led scientific team appeared February 10 in Nature, in an article entitled “Macromolecular diffractive imaging using imperfect crystals.” The article described how the scientists took advantage of a phenomenon called continuous diffraction.
Protein crystals, particularly imperfect protein crystals, do not always “diffract,” in the traditional Bragg sense. A proper, perfect crystal scatters X-rays in many different directions, producing an intricate and characteristic pattern of numerous bright spots, called Bragg peaks (named after the British crystallography pioneers William Henry and William Lawrence Bragg). The positions and strengths of these spots contain information about the structure of the crystal and of its constituents. Using this approach, researchers have already determined the atomic structures of tens of thousands of proteins and other biomolecules.
“Continuous” scattering arises when crystals become disordered. Usually, this non-Bragg continuous diffraction is not used to derive structural information. Instead, it is used to provide insights into vibrations and dynamics of molecules. But when the disorder consists only of displacements of the individual molecules from their ideal positions in the crystal, the “background” takes on a much more complex character—and its rich structure is anything but diffuse. It then offers a much bigger prize than the analysis of the Bragg peaks: The continuously modulated “background” fully encodes the diffracted waves from individual “single” molecules.
The possibility of using continuous diffraction for structural determinations leads to a paradigm shift in crystallography—the most ordered crystals are no longer the best to analyze with the novel method. “For the first time we have access to single molecule diffraction—we have never had this in crystallography before,” explained DESY’s Professor Henry Chapman. “But we have long known how to solve single-molecule diffraction if we could measure it.” The field of coherent diffractive imaging, spurred by the availability of laser-like beams from X-ray free-electron lasers, has developed powerful algorithms to directly solve the phase problem in this case, without having to know anything at all about the molecule.
“We show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly,” wrote the authors of the Nature article. “Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography.”