research advances
January 2009 research highlight
Paving the path to single-molecule structures
PSI-SGKB [doi:10.1038/nmeth0109-8a]
A new generation of brilliant X-ray laser sources will be coming online within the next few years. Researchers now show that using these lasers to determine the structures of single molecules should be possible.
The structure of the protein chignolin at 1.8 Å resolution, recovered from 72,000 simulated diffraction snapshots of unknown orientation. Reprinted from Nature Physics.
Obtaining the atomic structure of a protein by X-ray diffraction first requires purifying and crystallizing it. Although structural genomics efforts have laid pipelines for streamlining this tedious process, it still takes a great deal of time and effort, and many protein classes, such as membrane proteins, are still intractable to structure determination. A technology allowing researchers to obtain atomic structures of single protein molecules would eliminate the need for tiresome purification and crystallization, and would arguably have an enormous impact on structural biology.
Such a potentially revolutionary technology is not just a pipe dream. A new generation of X-ray sources known as X-ray free-electron lasers (XFELs) is being developed, and the first source, at the Stanford Linear Accelerator Center in the United States, is expected to be up and running as early as mid-2009. Researchers believe that these brilliant lasers, which come with up to a billion-dollar price tag, will facilitate single-molecule structure determination. “We think that these X-ray lasers are going to transform structure determination in the way that lasers transformed spectroscopy about 40 to 50 years ago,” says Abbas Ourmazd, a physicist at the University of Wisconsin, Milwaukee.
However, single-molecule structure determination will be a bit more complicated than just putting the molecule in the path of a powerful XFEL beam. The scattering of an X-ray signal by a single molecule is extremely faint, so many diffraction 'snapshots' must be collected to boost the signal. “Estimates are that one of these XFELs will deliver somewhere between 100 and 1,000 terabits of data per day, most of which will be zeros because single objects don't scatter very many photons,” explains Ourmazd. Thus a major question has been how to best use this sparse yet huge amount of data to reconstruct the three-dimensional structure of a single molecule.
Ourmazd and his co-workers found that the ideas proposed by other researchers to use XFEL data to solve single-molecule structures fell orders of magnitudes short. His group now has demonstrated, by applying algorithms that exploit the correlations between photons in a large series of X-ray scattering snapshots of a single object in random orientations, that they can recover a high-resolution atomic structure.
Ourmazd likens their method to reconstructing an ancient Greek vase from a pile of shards by maximizing the correlations between the pottery fragments. For reconstructing a molecular structure from a series of diffraction snapshots, the scattered photon arrangements are changed until the correlations between them are maximized. “Our technique depends primarily not on the number of photons you scatter per shot, but rather on the total number of photons you've scattered from all possible orientations of the object,” says Ourmazd. Using this correlation approach, his group reconstructed a high-resolution structure of the small protein chignolin from 72,000 simulated diffraction snapshots of the protein in random orientations at the expected scattered photon intensity. “Remarkably, the only information one needs is the dimensionality of the space in which the object lives,” he says.
This approach should theoretically allow researchers to obtain single-molecule structures for proteins of various sizes and even large ensembles such as viruses or nanoparticles. For looking at objects larger than chignolin, “the only requirement is to have a large enough computer,” notes Ourmazd. Looking at single molecules rather than bulk ensembles may also provide a route to mapping conformational heterogeneity, which is all but impossible with current crystallographic methods.
Though it will likely take several years of working out technical issues even after the first XFELs come online, it will certainly be very exciting to see whether the promise of single-molecule structure determination can be realized. “The proof of the pudding is in the eating, and there's nothing like taking real experimental data and showing everything works,” says Ourmazd. “We're looking forward to that.”
