research advances
February 2010 research highlight
One-shot structure determination
PSI-SGKB [doi:10.1038/nmeth0210-96a]
By sampling a two-dimensional diffraction pattern on a spherical detector, three-dimensional structure determination of single molecules should be possible from a single measurement.
The concept of ankylography. Left, a coherent beam illuminates a particle and the diffraction pattern is collected on a spherical surface (2θ max is the diffraction angle). Right, the 3D structure of a sodium silicate glass particle is encoded in the 2D spherical diffraction pattern. Reprinted from Nature.
The three-dimensional (3D) structure of an object can reveal a lot about its functional purpose. This is especially true at the microscopic scale, which is why many different methods have been developed to image biological particles, all the way down to the atomic level. To obtain 3D structures, however, one must reconstruct them from 2D snapshots. For a technique such as confocal microscopy, this involves sectioning biological specimens into planes and then assembling them back together to reconstruct the 3D image. For the technique of X-ray crystallography, this requires taking shots of multiple, identical copies of a biological molecule at different orientations, and reconstructing the 3D image from the diffraction patterns.
Although X-ray crystallography can yield atomic-resolution 3D structures of proteins and other biological molecules, it has two limitations. “The problem is that it requires crystals and also it [yields] average structures,” explains Jianwei Miao of the University of California, Los Angeles. Besides the challenge and time expense of crystallization, because X-ray crystallography is an 'averaging' technique, it cannot be used to look at single molecules. Thus the small structural differences between copies of a biomolecule, which are often crucial for understanding biological function, cannot be observed.
But now, Miao and his colleagues show that obtaining enough information to construct a 3D structure from a single diffraction measurement of a single particle should in principle be possible, using a new concept called ankylography.
The key idea of ankylography, which comes from the Greek word ankylos, meaning 'curved', is that a spherical (rather than planar) detector is used to record the diffraction pattern. A coherent X-ray beam is fired at the particle, and the diffraction pattern of the resulting scattered waves is recorded on a curved surface by, for example, a charge-coupled device (CCD) camera. If the diffraction pattern is sampled at a very fine scale, the 3D structure of the object is encoded by the 2D spherical pattern. Thus ankylography should allow the determination of 3D structures of single particles in one shot.
Using simulated diffraction data for a sodium silicate glass particle and for poliovirus, Miao and his colleagues demonstrated that this is possible, at least theoretically. They also imaged an etched silicon nitride substrate using experimental soft X-ray diffraction data, but because their CCD was equipped with a planar detector, they needed to mathematically interpolate what the pattern on a spherical detector would be.
To use ankylography in real-life experiments, additional technological developments are needed. For one, spherical detectors are needed. Also, robust computational algorithms to reconstruct 3D images from 2D spherical diffraction patterns will be necessary. Additionally, a new generation of ultrapowerful X-ray free electron lasers (XFELs), which are expected to transform the field of X-ray structure determination, are only just beginning to become available. These XFELs would be ideal to couple with ankylography; Miao predicts that 2-nanometer resolution from a single shot using an XFEL beamline would be possible, enabling 3D imaging of a broad range of biological specimens.
Technology development could potentially proceed rapidly. Hundreds of millions of dollars have already been invested for the development of XFELs and improved detectors in the US, Europe and Japan, says Miao, and many groups worldwide are working on the coherent imaging field using these advanced X-ray sources. “I think in a few years from now it should be much clearer what can be done with this technique,” he says. “This is a completely new idea so the community is still debating it; we've created a lot of discussion.”
