to BioTechniques free email alert service to receive content updates.
First Free-Electron Laser Structure

12/03/2012
Kelly Rae Chi

What molecule did researchers determine the structure of? And why did they select that target?


X-ray free-electron laser (XFEL) techniques have yielded their first new molecular structure. The analyzed molecule is a crucial enzyme of the parasite Trypanosoma brucei that causes the often-fatal human African trypanosomiasis (HAT) or sleeping sickness.

The structural data will inform the design of new inhibitors that target the parasitic rather than the human form of the enzyme, according to the study published online in Science on November 29 (1).

A map of intensities merged using the CrystFEL software suite from almost 200,000 diffraction patterns obtained from in vivo grown crystals of Trypanosoma brucei cathepsin B. This map is used to synthesize the 3D molecular structure of the enzyme. Image courtesy of Karol Nass, CFEL.





The detailed configuration of the molecule, cathepsin B, started with an accidental discovery by University of Tübingen graduate student Rudolph KoopmannKarol. He found that when expressed in insect cells, the parasite’s molecule forms small crystals—too small to be solved by conventional X-ray crystallography.

At the time, study author Henry Chapman, a founder of the Center for Free-Electron Laser Science at the Deutsches Elektronen Synchrotron (DESY) in Hamburg, Germany, was looking for a good system on which to demonstrate XFEL. His group has pioneered the technique for determining biological structures in unprecedented 3D detail (2,3). “I was asking around, ‘Does anyone have any little crystals?’” said Chapman.

XFEL works on similar principles as X-ray crystallography, a technique now generating structures for thousands of biomolecules each year. The success of X-ray crystallography, however, hinges on the ability to freeze proteins into large crystals. This is not always easy or possible.

In contrast, XFEL methods require smaller crystals. That’s because it uses a much brighter light source, so powerful that it can cut through steel and can easily vaporize a protein. With femtosecond pulses of exposure, the team can capture diffraction data before the molecules are destroyed. The eventual goal is eliminate the need for crystals completely, said Chapman.

In the new study, the team injected the XFEL with a steady flow of concentrated crystallized cathepsin B. They pulsed the laser four million times for a total of eight hours.

The experiment produced a staggering number of images. These images were analyzed with custom-designed algorithms to sort out whether pulses hit a crystal, and when they did, align the diffraction data from each randomly oriented crystal. A massive computer was built at DESY specifically to process the terabytes of data generated in the experiment.

With the structure of the unbound version of the enzyme in hand, the group found that the enzyme was crystallized in its natural state—with its native inhibitor. Their analysis uncovered differences between the binding sites of the parasite's and the human form of the protein.

Chapman’s group has been refining data processing, to be able to boost the speed of experiments. “Nowadays, we can almost solve structures while we’re at the beam line,” he said. Improvements to data processing will also reduce the required amount of concentrated protein.

The experiments were done using the first XFEL, the Linac Coherent Light Source, which was opened in 2009 at the SLAC National Accelerator Laboratory in Menlo Park, CA. A new-and-improved version of the laser, LCLS-II, with more powerful, seeded beams will allow scientists to solve even smaller crystals.

References

1. Redecke, L., K. Nass, D. P. DePonte, T. A. White, D. Rehders, A. Barty, F. Stellato, M. Liang, T. R. M. Barends, S. Boutet, G. J. Williams, M. Messerschmidt, M. M. Seibert, A. Aquila, D. Arnlund, S. Bajt, T. Barth, M. J. Bogan, C. Caleman, T.-C. Chao, R. B. Doak, H. Fleckenstein, M. Frank, R. Fromme, L. Galli, I. Grotjohann, M. S. Hunter, L. C. Johansson, S. Kassemeyer, G. Katona, R. A. Kirian, R. Koopmann, C. Kupitz, L. Lomb, A. V. Martin, S. Mogk, R. Neutze, R. L. Shoeman, J. Steinbrener, N. Timneanu, D. Wang, U. Weierstall, N. A. Zatsepin, J. C. H. Spence, P. Fromme, I. Schlichting, M. Duszenko, C. Betzel, and H. N. Chapman. 2012. Natively inhibited trypanosoma brucei cathepsin b structure determined by using an x-ray laser. Science (November) Epub ahead of print.

2. Miao, J, H.N. Chapman, J. Kirz, D. Sayre, and K.O. Hodgson. 2004. Taking X-ray diffraction to the limit: macromolecular structures from femtosecond X-ray pulses and diffraction microscopy of cells with synchrotron radiation. Annu Rev Biophys Biomol Struct. 33:157-76.

3. Chapman, H.N., P. Fromme, A. Barty, T.A. White, R.A. Kirian, A. Aquila, M.S. Hunter, J. Schulz, D.P. DePone, et al. 2011. Femtosecond X-ray protein nanocrystallography. Nature 470: 73-77.