R. Fung, A. Ourmazd, V. L. Shneerson, D. K. Saldin
X-ray crystallography is one of the key contributions of the physical sciences to the life sciences. Its application to biological, biochemical, and pharmaceutical problems continues to enable breakthroughs (e.g. Cramer et al., Science 292 1863, 2001) highlighting the importance of structure to function. However, roughly 40% of biological molecules do not crystallize, and many cannot easily be purified. These factors severely limit the applicability of X-ray crystallography; although more than 750,000 proteins have been sequenced, the structures of less than 10% have been determined to high resolution (Protein Data Bank, http://www.pdb.org). The ability to determine the structure of individual biological molecules – without the need for purification and crystallization – would constitute a fundamental breakthrough.
The confluence of five factors has generated intense interest in single-molecule crystallograph by short-pulse X-ray scattering: a) The advent of algorithms for determining phases from measured diffraction intensities by successive and repeated application of constraints in real and reciprocal spaces, with demonstrations in astronomy; diffractive imaging of nanoparticles, biological cells, small molecule crystallography, surface crystallography, and protein crystallography; b) Development of sophisticated techniques for determining the relative orientation of electron microscope images of biological entities, such as cells and large macromolecules; c) Development of techniques for producing beams of hydrated proteins by electrospraying or Raleigh-droplet formation; d) The promise of very bright, ultra-short pulses of hard X-rays from X-ray Free Electron Lasers (XFELs) under construction in the US, Japan, and Europe; e) The prospect of overcoming the limits to achievable resolution due to radiation damage by using short pulses of radiation.
It has been suggested (Neutze et al., Nature 406,752, 2000) that an experiment to determine the structure of a biological molecule might, in principle, proceed as follows: i) A train of individual hydrated proteins is exposed to a synchronized train of intense X-ray pulses. As a single pulse is sufficient to destroy the molecule, the pulses (and data collection) must be short compared with the roughly 50 fs needed for the molecular constituents to fly apart. ii) The two-dimensional (2D) diffraction patterns obtained with single pulses are read out, each pattern corresponding to an unknown, random orientation of the molecule. iii) The relative orientations of the molecule corresponding to 2D diffraction patterns (and hence the relative orientations of each diffraction pattern in 3D reciprocal space) are determined. iv) A noise-averaged 3D diffracted intensity distribution is constructed. v) The structure of the molecule is determined from the diffracted intensity distribution by an iterative “phasing algorithm”.
We have recently succeeded in reconstructing the electron density of the small synthetic protein chignolin (1UAO) from diffraction patterns of randomly oriented molecules of a a signal level expected of an x-ray free electron laser (XFEL). If your browser does not automatically display this electron density in this page, click here to see the .avi movie in a movie player on your computer.