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.