User Consortium of Serial Femtosecond Crystallography - Slovak Involvement

I. Barák¹, K. Saksl² and P. Sovák³

¹Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21,

845 51 Bratislava 45, Slovakia

²Institute of Materials Research, Slovak Academy of Sciences, Watsonova 47,

040 01 Košice, Slovakia

³Department of Condensed Matter Physics, P.J. Šafárik University, Park Angelinum 9,

040 01Košice, Slovakia

Structure determination at synchrotrons often follows years of tremendous effort to grow large protein crystals that are well enough ordered to diffract to high resolution within the limited exposure to avoid radiation damage. There are no guarantees that the crystal observed under the polarizing microscope, and which appears to be a “good” crystal, will actually diffract.

X-ray free-electron lasers are opening up unique opportunities to image biological materials at high resolution. The high-intensity ultra-short pulses provided by these sources enable us to overcome radiation damage limits by outrunning the structural degradation that inevitably occurs on exposure of soft matter to ionizing radiation [1]. The experience at LCLS (Linac Coherent Light Source, Stanford, USA) showed that most nano- and microcrystalline protein samples that have been tried have diffracted, and usually to higher resolution than has been observed from synchrotron studies on macroscopic crystals, in test cases where these large crystals were available. High-resolution diffraction has been obtained from integral membrane proteins, including G-protein coupled receptors, in the form of sub-micron crystals carried in a lipidic cubic phase matrix. LCLS produces pulses of X-rays more than a billion times brighter than the synchrotron sources which are also based on large electron accelerators.

X-ray free-electron lasers are opening up unique opportunities to image biological materials at high resolution. The high-intensity ultra-short pulses provided by these sources enable us to overcome radiation damage limits by outrunning the structural degradation that inevitably occurs on exposure of soft matter to ionizing radiation [1]. This central idea makes many new techniques possible for the study of reproducible (and quasi-reproducible) structures such as macromolecules and viruses. These methods include single-particle diffractive imaging of viruses [2], and the extension of protein crystallography to smaller and smaller crystal sizes (avoiding the years of development often required to grow large crystals) [3]. All current evidence points to the fact that serial femtosecond crystallography (SFX) could have a remarkable and profound impact on the field of structural biology. Not only does SFX overcome the crystallization bottleneck, but data can be collected very rapidly (several minutes to collect about 10 000 images for complete 3D atomistic reconstruction) and over a wide range of temperatures. Indeed, measurements can most easily be carried out at room temperature. In conventional synchrotron-based macromolecular crystallography, samples are usually frozen to reduce radiation damage, and the crystallization process usually, but not always, allows only a single protein conformation to be studied. The results from other techniques, such as cryo-electron microscopy and atomic force microscopy, make it increasingly clear that this shortcoming of MX is limiting our view of protein interactions. Recent macromolecular crystallography studies at room temperature has shown that flash cooling to reduce radiation damage can bias hidden structural ensembles in protein crystals and remodel the conformational distribution of 35% of side-chains, while eliminating the packing defects necessary for functional motions. Thus room temperature measurements can reveal motions crucial for catalysis, ligand binding and allosteric regulation [4].

SFX technique is also a natural fit for time-resolved measurements at femtosecond timescales. Despite valuable progress in the time-resolved protein crystallography, what is urgently needed is a time-resolved technique which can image individual proteins at sub-nanometer resolution in three dimensions, in their native environment, unaffected by damage from the imaging radiation. Only this technique offers this possibility.

The European XFEL brings a unique capability of over a 200 increase in pulse repetition rate compared with the LCLS, which will vastly increase the efficiency of the method, reducing the time required to carry out a measurement and reducing the quantity of protein required to obtain a structure. The potential user community for SFX is extremely large, and encompasses much of the user base for synchrotron macromolecular crystallography stations around the world. In fact the user base is potentially much larger than that, since this method vastly increases the number of samples that now can be analyzed. Taken together, X-ray FELs have the potential to profoundly impact the field of structural biology. The Slovak scientists are ready to participate in this exciting initiative. There is a plan for the structural biology community participation, specifically to set up a protein quality pre-screening centre at IMB SAS. Further Slovak involvement in User consortium of SFX at XFEL experimental station in Hamburg will be discussed.

[1] R. Neutze, R. Wouts, D. van der Spoel, E. Weckert, and J. Hajdu. Potential for biomolecular imaging with femtosecond x-ray pulses. Nature: 406:753–757, 2000.

[2] M. M. Seibert, T. Ekeberg, F. R. N. C. Maia, M. Svenda, J. Andreasson,O. Jonsson, D. Odic, B. Iwan, A. Rocker, D. Westphal, et al. Single mimivirus particles intercepted and imaged with an x-ray laser. Nature: 470:78–81, 2011.

[3] H. N. Chapman, P. Fromme, A. Barty, T. A. White, R. A. Kirian, A. Aquila, M. S. Hunter, J. Schulz, D. P. DePonte, U. Weierstall, et al. Femtosecond x-ray protein nanocrystallography. Nature: 470:73–77, 2011.

[4] Fraser JS, van den Bedem H, Samelson AJ, Lang PT, Holton JM, Echols N, Alber T. Accessing protein conformational ensembles using room-temperature Xray crystallography Proc. Nat. Acad. Sci. USA 108:16247, 2010.