In two Letters in Nature, scientists are reporting that they have been able to reconstruct the structures of biological molecules using femtosecond X-ray pulses. In the first Letter, they imaged nano-crystals of the photosystem I protein, part of the photosynthetic machinery. In the second, they were able to image individual single mimiviruses, part of a family of giant viruses. We seem to be well on the way to single molecule structural imaging.

X-ray diffraction crystallography has been around since the 1920s and is a useful tool for determining the structures of molecules. The basic idea is that the photons diffract as they strike the ordered structure of crystals. The resulting diffraction pattern is unique to the structure of the crystal. So, a crystal of a protein creates a unique diffraction pattern that tells us where the protein's atoms reside.

Although many molecular structures have been solved, the technique is far from perfect. For one, not all molecules crystallize well or form sufficient sized crystals for current techniques. Worse, the protein may crystallize in a nonfunctional form. So, being able to work with smaller crystals or, better yet, single molecules, would be a huge advantage.

One team has created a stream of fluid containing nano-crystals of the photosystem I protein (the crystals are a few hundred nanometers in diameter). The nano-crystal stream is hit with a 1.8 keV X-ray beam that is focused down to a 7 µm spot and pulsed at 30 Hz. Each pulse shot lasts between 10-200 femtoseconds and exceeds power densities of 1016 W cm-2. Yes, that’s 10,000 trillion watts per square cm. Normal X-ray crystallography uses a fraction of that power (we described the construction of this X-ray source last year).

In these experiments, the nano-crystals are completely destroyed, but not before they create a sufficiently bright diffraction pattern. And, at 30 Hz, that is 1,800 patterns per minute. By processing thousands of patterns, the team was able to confirm the crystal structure of the photosystem I protein with a resolution of 8.5 Angstroms. That's not as good as the best traditional methods, but it required a lot less starting material.

The second group was able to produce a diffraction pattern from a non-crystallized biological sample, a Acanthamoeba polyphaga mimivirus (diameter ~0.75 µm). A stream of buffer and viruses was injected into the X-ray beam line, and hit with pulses 10 µm in diameter with a power density of 6.5x1015 W cm-2. Before the mimivirus "explodes and turns into plasma," enough photons were collected to image a diffraction pattern. From the diffraction pattern, they were able to reconstruct the capsid of the virus accurately with a resolution of 32 nm. This is very impressive because the diffraction pattern and reconstruction came from a single exposure of a single particle.

The teams have successfully demonstrated that they can get sufficient diffraction signal from nano-crystals or individual macro-molecules before the samples are destroyed by the intense illumination. In both cases, higher energy x-rays and shorter bursts are expected to increase resolution and decrease the size of the sample particles. Plans are already in the works for x-ray lasers 105 times brighter but with pulses lasting less than 10fs.

Nature, 2011. DOI: 10.1038/nature09750, 10.1038/nature09748 (About DOIs).

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