New Method Opens Crystal Clear Views of Biomolecules

Slightly disordered crystals of complex biomolecules like that of the photosystem II molecule shown here produce a complex continous diffraction pattern (right) under X-ray light that contains far more information than the so-called Bragg peaks of a strongly ordered crystal alone (left). The degree of disorder is greatly exaggerated in the crystal on the right. (Source: E. Reimann, DESY)

Slightly disordered crystals of complex biomolecules like that of the photosystem II molecule shown here produce a complex continous diffraction pattern (right) under X-ray light that contains far more information than the so-called Bragg peaks of a strongly ordered crystal alone (left). The degree of disorder is greatly exaggerated in the crystal on the right. (Source: E. Reimann, DESY)

A scientific breakthrough gives researchers access to the blueprint of thousands of molecules of great relevance to medicine and biology. The novel technique, pioneered by a team led by DESY scientist Henry Chapman from the Center for Free-Electron Laser Science CFEL opens up an easy way to determine the spatial structures of proteins and other molecules, many of which are practically inaccessible by existing methods. The structures of biomolecules reveal their modes of action and give insights into the workings of the machinery of life. Obtaining the molecular structure of particular proteins, for example, can provide the basis for the development of tailor-made drugs against many diseases. “Our discovery will allow us to directly view large protein complexes in atomic detail,” says Chapman, who is also a professor at the University of Hamburg and a member of the Hamburg Centre for Ultrafast Imaging CUI.

To determine the spatial structure of a biomolecule, scientists mainly rely on crystallography. But crystallography suffers from two significant barriers, which make structure determination extremely difficult or sometimes impossible. The first is that the molecules must be formed into very high quality crystals. Most biomolecules do not naturally form crystals. However, without the necessary perfect, regular arrangement of the molecules in the crystal, only a limited number of Bragg peaks are visible and the structure cannot be determined.

The second barrier is that the structure of a complex molecule is still extremely difficult to determine, even when good diffraction is available. “This task is like extreme Sudoku in three dimensions and a million boxes, but with only half the necessary clues,” explains Chapman. In crystallography, this puzzle is referred to as the phase problem. Without knowing the phase it is not possible to compute an image of the molecule from the measured diffraction pattern. Since phases can’t be measured more information must be known than just the measured Bragg peaks. This additional information can sometimes be obtained by X-raying crystals of chemically modified molecules, or by already knowing the structure of a closely-related molecule.

The analysis of the Bragg peaks alone (top) reveals far less details than the analysis of the continuous diffraction pattern (bottom). Magnifying glasses show real data. (Source: E. Reimann, DESY)

The analysis of the Bragg peaks alone (top) reveals far less details than the analysis of the continuous diffraction pattern (bottom). Magnifying glasses show real data. (Source: E. Reimann, DESY)

When thinking about why protein crystals do not always “diffract”, Chapman realized that imperfect crystals and the phase problem are linked. The key lies in a weak “continuous” scattering that arises when crystals become disordered. Usually, this non-Bragg, continuous diffraction is thought of as a nuisance, although it can be useful for providing 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 then 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.

With imperfect, disordered crystals, the continuous diffraction fills in the gaps and beyond the Bragg peaks, giving vastly more information than in normal crystallography. With this additional gain in information, the phase problem can be uniquely solved without having to resort to other measurements or assumptions.

The new work offers a direct route to “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,” says the chairman of DESY’s board of directors, Helmut Dosch. 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. “You don’t even have to know chemistry,” says Chapman, “but you can learn it by looking at the three-dimensional image you get.”

Including the continuous diffraction pattern into the analysis immediately improved the spatial resolution around a quarter from 4.5 to 3.5 Ångström. The obtained image gave fine definition of molecular features that usually require fitting a chemical model to see. “That is a pretty big deal for biomolecules,” explains Anton Barty from DESY. “And we can further improve the resolution if we take more patterns.”

The scientists hope to obtain even clearer and higher resolution images of photosystem II and many other macromolecules with their new technique. “This kind of continuous diffraction has actually been seen for a long time from many different poorly-diffracting crystals,” says Chapman. “It wasn’t understood that you can get structural information from it and so analysis techniques suppressed it. We’re going to be busy to see if we can solve structures of molecules from old discarded data.” (Source: DESY)

Link: Coherent Imaging Division (Prof. Chapman), Center for Free-Electron Laser Science, CFEL, Deutsches Elektron-Synchrotron (DESY), Hamburg, Germany

Reference: K. Ayyer et. al: Macromolecular diffractive imaging using imperfect crystals, Nature 530; doi:10.1038/nature16949

Speak Your Mind

*