Tables for
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 1.4, pp. 41-42

Section 1.4.4. Gazing into the crystal ball – the X-ray free-electron laser (J. C. H. Spence)

E. Arnold,a* M. G. Rossmann,b D. M. Himmel,a J. C. H. Spencec and S. Sunb

aBiomolecular Crystallography Laboratory, CABM & Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854–5638, USA,bDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907–1392, USA, and cDepartment of Physics, Arizona State University, Tempe, Arizona, 85287, USA
Correspondence e-mail:

1.4.4. Gazing into the crystal ball – the X-ray free-electron laser (J. C. H. Spence)

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The recent invention of the pulsed hard X-ray (free-electron) laser (XFEL) is certain to impact structural biology, particularly in the areas of protein nanocrystal analysis (Chapman et al., 2011[link]), single-particle imaging (Siebert et al., 2011[link]), time-resolved crystallography and solution scattering (see the forthcoming reviews in Reports on Progress in Physics by Spence and Chapman). Current hard-X-ray machines provide about 1012 photons in each 40 femtosecond pulse, and are capable of reading out perhaps 120 of the resulting diffraction-pattern `snapshots' every second. Such a beam, focused to micron dimensions, vaporizes the sample, but it has been discovered that a useful pattern is obtained before radiation damage commences (due to the photoelectron cascade). The method has given 2 Å resolution data from micron-sized protein nanocrystals, and, if sufficiently brief pulses are used, allows about 100 times greater dose to be delivered than the Henderson `safe dose' (see Chapter 10.3[link] ). The snapshot data consist of partial reflections. As a consequence of the fully coherent nature of the radiation, for the smallest submicron nanocrystals the data show interference fringes between the Bragg reflections that facilitate iterative phasing (Spence et al., 2011[link]). Sample delivery has been based on a continuously flowing liquid jet of micron or submicron dimensions, freely flowing in vacuum, with gas focusing at a nozzle to prevent clogging. Merging of millions of nanocrystal snapshots to obtain full reflections has created new challenges for data analysis, as has the development of MAD phasing for the time-resolved absorption involved.

Diffraction patterns may also be obtained from single particles such as a virus or whole cell, commonly injected from a nebulizer in a gas-focused stream into vacuum. Each snapshot gives one projection of the particle in a random orientation, so that three-dimensional reconstruction requires a solution to the difficult problems of orientation determination and phasing of single-particle diffraction patterns. The available X-ray fluence per shot, together with the minimum amount of scattering needed for orientation determination, has so far limited resolution to about 30 nm. However, more powerful X-ray lasers, smaller focused spots and improvements in `hit rate' are bound to improve resolution to the predicted 1 nm resolution limit for particles too thick for study by cryogenic electron microscopy (cryo-EM). Scattering in the water window, around 500 eV, gives greatly increased protein/water contrast, but wavelength-limited resolution. Conformational variability imposes similar limitations to those encountered in cryo-EM, and the merging of multiple projections from similar cells remains an important challenge for the future, perhaps based on topological constraints. The XFEL also offers unprecedented opportunities for time-resolved imaging, spanning the range from the femtosecond timescale important for electron-transfer reactions in biochemistry, to the slower microsecond processes of protein activity. In favourable cases, a fast optical trigger exists for pump–probe studies, while in others chemical reactions (such as the enzyme cycle) might be followed in mixed and flowing solutions. For this purpose, the correlated fluctuations in `snapshot' small-angle X-ray (SAX) patterns may prove useful, since they offer a hit rate of 100%. Since these patterns are two-dimensional for particles frozen in space or time, they contain more information than conventional one-dimensional SAX patterns. The ability to reconstruct an image of one particle using the scattering from many randomly oriented particles frozen in space (without modelling) has recently been demonstrated (Saldin et al., 2011[link]). In summary, the XFEL has opened up many new exciting possibilities for structural and dynamic biology, based on entirely new experimental arrangements (now far from optimized) and offering great scope for developments in this highly interdisciplinary field, which spans laser, detector and particle injector physics, diffraction physics, and structural biology. We anticipate rapid progress in methods for the growth of suitable protein microcrystals, especially for membrane proteins. The XFEL is then certain to provide a wealth of new information on molecular mechanisms in biology, as techniques are refined and more powerful X-ray lasers are constructed.


Chapman, H. N., Fromme, P., Barty, A., White, T. A., Kirian, R. A., Aquila, A., Hunter, M. S., Schulz, J., DePonte, D. P., Weierstall, U., Doak, R. B., Maia, F. R., Martin, A. V., Schlichting, I., Lomb, L., Coppola, N., Shoeman, R. L., Epp, S. W., Hartmann, R., Rolles, D., Rudenko, A., Foucar, L., Kimmel, N., Weidenspointner, G., Holl, P., Liang, M., Barthelmess, M., Caleman, C., Boutet, S., Bogan, M. J., Krzywinski, J., Bostedt, C., Bajt, S., Gumprecht, L., Rudek, B., Erk, B., Schmidt, C., Hömke, A., Reich, C., Pietschner, D., Strüder, L., Hauser, G., Gorke, H., Ullrich, J., Herrmann, S., Schaller, G., Schopper, F., Soltau, H., Kühnel, K. U., Messerschmidt, M., Bozek, J. D., Hau-Riege, S. P., Frank, M., Hampton, C. Y., Sierra, R. G., Starodub, D., Williams, G. J., Hajdu, J., Timneanu, N., Seibert, M. M., Andreasson, J., Rocker, A., Jönsson, O., Svenda, M., Stern, S., Nass, K., Andritschke, R., Schröter, C. D., Krasniqi, F., Bott, M., Schmidt, K. E., Wang, X., Grotjohann, I., Holton, J. M., Barends, T. R., Neutze, R., Marchesini, S., Fromme, R., Schorb, S., Rupp, D., Adolph, M., Gorkhover, T., Andersson, I., Hirsemann, H., Potdevin, G., Graafsma, H., Nilsson, B. & Spence, J. C. (2011). Femtosecond X-ray protein nanocrystallography. Nature (London), 470, 73–77.
Saldin, D. K., Poon, H. C., Bogan, M. J., Marchesini, S., Shapiro, D. A., Kirian, R. A., Weierstall, U. & Spence, J. C. H. (2011). New light on disordered ensembles: Ab-initio structure determination of one particle from scattering fluctuations of many copies. Phys. Rev. Lett. 106, 115501.
Siebert, M. M. et al. (2011). Single mimivirus particles intercepted and imaged with an X-ray laser. Nature (London), 470, 78–81.
Spence, J. C., Kirian, R. A., Wang, X., Weierstall, U., Schmidt, K. E., White, T., Barty, A., Chapman, H. N., Marchesini, S. & Holton, J. (2011). Phasing of coherent femtosecond X-ray diffraction from size-varying nanocrystals. Opt. Express, 19, 2866–2873.

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