Feb 11 2016
Forming the high-quality crystals required for X-ray analysis of the structure of biological molecules is often the most difficult part of taking atomic-resolution images. Using the world's brightest X-ray source, at the Department of Energy's SLAC National Accelerator Laboratory, researchers have demonstrated that sharp images are obtainable, even with imperfect crystals.
Artistic representation of an imperfect crystal, in which individual repeating units (represented here by ducks rather than biomolecules) are displaced from their ideal positions on a regular crystal lattice (tiles). In X-ray crystallography, researchers shine X-rays through crystals to determine the atomic structure of the crystals' units, such as complex biological molecules. A new study of imperfect crystals at SLAC's LCLS X-ray laser has shown that the imperfections can be exploited to obtain much higher-resolution images than with conventional methods. (Credit: SLAC National Accelerator Laboratory.)
The amazing results have the potential to eliminate the search for better crystals, and could essentially change the way in which researchers are studying the biological machinery that underlie catalysis, photosynthesis and other vital processes of living organisms. A better knowledge about such processes could pave the way for innovations in many fields, such as clean energy production and drug development.
Once the full potential of the new method is understood, it could turn out to be one of the biggest advances since the birth of crystallography.
Mike Dunne, Director of the Linac Coherent Light Source (LCLS) X-ray laser, a DOE Office of Science User Facility
The new findings were published in Nature.
Signals Between Signals
Australian-born, British physicist William Lawrence Bragg discovered a method over 100 years ago using X-rays to probe crystal interiors, which includes arrays of atoms or molecules. This discovery lead the way for X-ray crystallography, an important technique to analyze material structures, biological molecules, and chemical processes.
X-ray crystallography has been very successful in determining proteins atomic structures and protein function. It has helped to determine more than 100,000 protein structures, which requires multiple copies of a protein for incorporation into a single crystal.
X-rays disperse the protein molecules and form a diffraction pattern on a detector, while passing through a crystal. This pattern consists mainly of bright spots referred to as Bragg peaks, which are used to reconstruct the molecules’ atomic structures.
Only Bragg peaks are produced in a perfectly ordered crystal. However the number of peaks, and the molecular image resolution obtainable from the peaks, can be limited if disorder exists.
Between two Bragg peaks and beyond them, gentle rippling patterns are produced due to disorder. Iit was thought that the patterns are not able to produce high-resolution molecular images, although the patterns, referred to as “continuous diffraction,” have been considerably researched.
We've now demonstrated that we can actually use the continuous diffraction of imperfect crystals to obtain better molecular images than with Bragg peaks alone.
Kartik Ayyer, The Center for Free-Electron Laser Science, DESY
The method was employed by the researchers to photosystem II; a large protein machine engaged in photosynthesis. It was found that merging the information from Bragg signals and non-Bragg signals offered improved high-resolution images, with better structural details compared to the images acquired by the traditional Bragg-only process.
Crystallography Meets Single-Particle Imaging
It was evident that the continuous diffraction of the crystals originates from the crystal lattice molecules that have moved out of their ideal locations by an amount as minute as the width of an atom. X-rays scattering off these shifted molecules merge to create the noticeable continuous pattern instead of Bragg peaks.
We already know how to analyze these signals. What's special about the continuous diffraction is that it contains significantly more information about the molecular structure than can possibly be measured using Bragg peaks alone. This completely changes our ability to determine the structures of these large, complex biological machines from an almost impossible task to a solvable problem.
Henry Chapman, The Center for Free-Electron Laser Science, DESY
In principle this technique permits researchers to rebuild a biomolecule’s atomic structure from the beginning, without knowing beforehand the positions of some of the atoms or the structure of an identical protein, eliminating a major problem.
The technique is a very elegant marriage between two approaches: X-ray diffraction of crystals and X-ray imaging of single particles. It uses the best of both worlds.
Ilme Schlichting, German Max Planck Institute for Medical Research
The method could be the first step towards molecular imaging of single particles, one of the key objectives in modern X-ray science. The approach permits measuring several biological samples, which are difficult to crystallize. Determining individual molecular orientations is another challenge to overcome, as diffraction intensities of single molecules are very weak. Obtaining several copies of the same molecule in the crystal lattice of imperfect crystals solves the two problems.
New Approach with Promising Perspectives
The method promises to alter the manner in which scientists use X-ray lasers for biological research, with its wider applications also being evaluated. It is not yet known whether the method could be employed at synchrotron facilities, where X-ray sources are more widespread than X-ray lasers though less powerful.
Since light from LCLS is so bright, our data could be taken very rapidly and on very small crystals. The same experiment at a synchrotron is likely to be more challenging because it would require longer exposures to X-rays, increasing the risk for sample damage, and also require larger crystals that are more likely to show additional unwanted disorder.
Sébastien Boutet, LCLS researcher
The researchers feel that the method could work for other biomolecules besides photosystem II.
The kind of disorder used in this research occurs frequently. It makes the approach an extremely valuable tool.
Ilme Schlichting, German Max Planck Institute for Medical Research
Besides DESY and SLAC, other institutions also contributed to the study, namely, University of Hamburg and the Center for Ultrafast Imaging in Germany; Arizona State University; the University of Wisconsin, Milwaukee; and the Foundation for Research and Technology-Hellas in Greece. The study received support from the Helmholtz Association, Germany; the Deutsche Forschungsgemeinschaft, Germany; the European Research Council; the Federal Ministry of Education and Research of Germany; the University of Hamburg, Germany; the BioXFEL Science Technology Center; the U.S. National Institutes of Health, National Institute of General Medical Sciences; and the DOE Office of Science, Office of Basic Energy Sciences.