|A New Light on Disordered Ensembles|
Because individual biomolecules are very small, x-ray scattering experiments usually determine their structures by an analysis of scattering from a large number of them. In crystallography, scattering by many molecules in identical orientations vastly enhances the signal from a single molecule. However, not all biomolecules form crystals. They are more usually found in disordered ensembles in aqueous solutions or in biomembranes. Now, researchers from Arizona State University, SLAC National Accelerator Center, Berkeley Lab, Brookhaven National Laboratory, and the University of Wisconsin-Milwaukee have performed, at ALS Beamline 9.0.1, the first experimental demonstration of a method that amplifies the information in the x-rays that scatter from disordered biomolecules, allowing the reconstruction of an image of a single molecule from fluctuations in the scattering from an ensemble of randomly oriented copies.
The overwhelming majority of known molecular structures at the atomic scale have been determined by x-ray crystallography, a technique that cannot be applied to molecules that resist crystallization (e.g., many membrane proteins). One solution to this problem would be to exploit the billionfold increase in peak brightness provided by an x-ray free-electron laser, due to which it may be possible to detect meaningful signals from single microscopic particles. Because of the large flux of radiation, however, it is necessary to work in the so-called "diffract and destroy" mode, in which the x-ray pulse must terminate within about 50 fs to avoid resolution-limiting effects resulting from the disintegration of the particle. The difficulty of targeting a single particle in such an experiment could be overcome with the development of a method for extracting structural information from scattering by a disordered ensemble of particles.
The absence of periodicity in such a sample means that the scattered x-rays form a continuous distribution, as opposed to discrete Bragg peaks. This allows for analysis of the data at a finer sampling rate, both radially (outward from the center) as well as angularly (around a ring of constant radius). If sampled finely enough, it has been suggested that enough information to reconstruct an image may be obtained from minute fluctuations in the angular intensities.
In these experiments, the particles were nanorods varying in length and diameter by approximately 10% to 15% in both dimensions; a small but significant fraction (~20%) of the nanoparticles were spherical in shape. The sizes of the nanoparticles were approximately that of a typical virus, suggesting a possible application to determining the structures of virus particles supported on a similar substrate. The researchers collected soft x-ray transmission diffraction patterns using 750-eV highly coherent x-rays (1.65 nm in wavelength). Hundreds of diffraction patterns were collected from different 15-micron-diameter regions, each containing approximately 10 ± 5 nanorods. The experiments also showed that, even if the measured diffraction patterns are from two different particle shapes (in this case a nanorod and a nanosphere), simultaneous reconstruction of the real-space structures of the both particle types is possible.
As demonstrated for the first time in these experiments, the advantages of signal amplification, damage reduction, and access to oversampled intensities may be combined to determine the structure of a single particle by diffraction patterns from many identical particles with neither translational nor orientational order. The extra information present in the angular correlations allows for an ab initio reconstruction, free of modeling and a priori assumptions.
Research conducted by R.A. Kirian, U. Weierstall, and J.C.H. Spence (Arizona State University); M.J. Bogan (SLAC National Accelerator Laboratory); S. Marchesini (ALS); D.A. Shapiro (Brookhaven National Laboratory); and H.C. Poon and D.K. Saldin (University of Wisconsin-Milwaukee).
Research funding: U.S. Department of Energy (DOE), Office of Science, and the National Science Foundation. Operation of the ALS is supported by the DOE Office of Basic Energy Sciences.
Publication about this research: D.K. Saldin, H.C. Poon, M.J. Bogan, S. Marchesini, D.A. Shapiro, R.A. Kirian, U. Weierstall, and J.C.H. Spence, "New light on disordered ensembles: Ab initio structure determination of one particle from scattering fluctuations of many copies," Phys. Rev. Lett. 106, 115501 (2011).
ALS Science Highlight #226