Abstract
It has been suggested that the extended intensity profiles surrounding Bragg reflections that arise when a series of finite crystals of varying size and shape are illuminated by the intense, coherent illumination of an x-ray free-electron laser may enable the crystal’s unit-cell electron density to be obtained ab initio via well-established iterative phasing algorithms. Such a technique could have a significant impact on the field of biological structure determination since it avoids the need for a priori information from similar known structures, multiple measurements near resonant atomic absorption energies, isomorphic derivative crystals, or atomic-resolution data. Here, we demonstrate this phasing technique on diffraction patterns recorded from artificial two-dimensional microcrystals using the seeded soft x-ray free-electron laser FERMI. We show that the technique is effective when the illuminating wavefront has nonuniform phase and amplitude, and when the diffraction intensities cannot be measured uniformly throughout reciprocal space because of a limited signal-to-noise ratio.
- Received 31 July 2014
DOI:https://doi.org/10.1103/PhysRevX.5.011015
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Published by the American Physical Society
Popular Summary
The vast majority of high-resolution biological macromolecule structures have been determined using x-ray crystallography. However, the well-known crystallographic “phase problem” forbids the determination of most structures without prior-known structural information or additional (and more challenging) diffraction measurements. We demonstrate an effective means of using exceptionally brief and intense coherent x-ray pulses from free-electron lasers to overcome the crystallographic phase problem without the need for prior structural knowledge, resonant conditions, or modifications to the molecular structures. This method applies to diffraction patterns recorded over a period shorter than that of atomic motions, which reveals undamaged states of microcrystals at physiological temperatures.
In the 1950s, David Sayre postulated that a general and direct solution to the crystallographic phase problem could be conceived if the diffraction intensities between Bragg reflections could somehow be measured. This observation led to the development of coherent diffractive imaging for noncrystalline targets, yet the crystallographic measurements that Sayre called for have largely eluded experimentalists. This situation persisted until 2009, when the first femtosecond protein nanocrystallography experiments that were performed using free-electron lasers revealed protein-crystal diffraction between Bragg reflections. Our proof-of-principle experiment, performed on two-dimensional platinum microcrystals, utilizes 32.5-nm x rays from the FERMI free-electron laser. We provide an experimental demonstration of how the continuum of diffraction intensities resulting from coherently illuminated microcrystals can be utilized to yield crystallographic phases and thus the crystal unit-cell density.
Our method, which places no restriction on diffraction resolution, bridges the divide between the phase problems associated with continuous Fruanhofer diffraction and crystalline Bragg diffraction. It is particularly well suited to the rapidly evolving field of serial femtosecond crystallography, which has recently been shown to be an efficient means of obtaining atomic-resolution structures and overcomes problems associated with small microcrystals, radiation damage, time resolution, and cryogenic freezing.
