Attosecond Serial Crystallography

Very recently, our collaborators and we have introduced a new paradigm of femtosecond serial nano crystallography for collecting protein diffraction data from sub-micron crystals with ~10 to 100-fs pulses from X-ray FELs [Chapman 2011, Redecke 2012, Boutet 2012]. Our method combines two major advances: Firstly, the short exposure allows for diffraction patterns of the crystals be collected before the destruction occurs, even though the degree of ionization and subsequent Coulomb explosion is extreme [Neutze 2000, Barty 2012]. “Damage-free” data have been collected at doses exceeding 3 GGy, from room-temperature samples. This exceeds 3000 times the tolerable dose for conventional crystallography at room temperature and is 100 times higher than the tolerable dose for cryogenically cooled samples. Secondly, we merge data from hundreds of thousands of micron or sub-micron crystals that flow continuously across the X-ray beam as a liquid suspension in a micro-jet [Weierstall 2012, DePonte 2008]. Each pulse gives rise to a unique snapshot diffraction pattern of a randomly-oriented crystal. Diffraction data are combined for each indexed Bragg spot by Monte Carlo integration, [Kirian 2010]. This merging of terabytes of data [White 2012] accumulates signals from many individual crystallites. We achieve a full 3D dataset that can be analyzed by conventional single-crystal phasing techniques, including anomalous diffraction [Son 2011]. Both of these advances allow us to use protein crystal sizes that would be prohibitively small for conventional studies, even with the brightest synchrotron sources. Consequently, we overcome the major bottleneck of structural biology, which is the requirement for large well-diffracting crystals that can take years of effort to achieve. Furthermore reactions can be induced in the small crystals by light or rapid mixing “on the fly”, which allows for time resolved data to be collected [Aqulia 2012, Kupitz 2014, Tenboer 2014].

The method of serial crystallography with X-ray FEL pulses has already been applied to some of the most difficult protein complexes, including membrane protein complexes such as G-protein coupled receptors [Liu 2013, Kang 2015, Zhang 2015], and Photosystem I and II (see Fig. 1) [Chapman 2011, Kupitz 2014]. Since the sample is refreshed on every pulse, this method is also ideal for time-resolved measurements of irreversible reactions [Aquila 2012] with an inherently high temporal resolution.

In the AXSIS project, we will push the technique of serial crystallography to a new frontier and overcome limitations of current fs FELs, using attosecond pulses from the C-ICS source. Although fs pulses from FELs are indeed short enough to collect X-ray diffraction data before the molecule is destroyed [Barty 2012], the ionization and destruction of electronic states in molecules is particularly severe [Rudek 2012]. As shown by electron spectroscopy and fluorescence [Young 2010, Rudek 2012], core electrons are rapidly displaced and “hollow atoms” created. On large molecules and crystals the fastest loss is due to impact ionization due to photoelectrons liberated and Auger decay which requires attosecond pulses to outrun [Son 2011]. By bringing the technique from the X-ray FEL to a compact laboratory-scale X-ray source we vastly extend its availability. Attosecond pulses from the C-ICS source, while having fewer photons than the large scale XFEL (yet matching the peak power of those sources), are spectrally better defined and fully longitudinal coherent and will thereby provide X-ray diffraction patterns that can be more easily interpreted and merged, giving further gains in accuracy and reduce sample consumption. Furthermore, the electronic structure is not disturbed by the attosecond pulses, which allows for truly damage-free X-ray spectroscopy to be performed. A new advanced CMOS pixel array detector will be built to match the 1-kHz repetition rate of the source and to meet the dynamic range requirements. We will develop an X-ray emission and absorption spectroscopy (XES, XAS) setup [Alonso-Mori 2012a, Alonso-Mori 2012b, Davis 2012], which will be combined with X-ray diffraction for simultaneous collection of XES/XAS spectra and diffraction data at 1-kHz repetition rate.

The groundbreaking experimental capabilities of attosecond X-ray diffraction and spectroscopy will be used to realize one of the grandest dreams of chemists and biochemists: the production of molecular movies of the formation of the excited states and transition states of molecules at the atomic level. By adding optical and X-ray spectroscopies we will gain new insights in spectroscopic measurements by giving a real-space accompaniment to changes in electronic states. Using this capability we will unravel the mechanisms of ultrafast light absorption and excitation energy transfer in important biological processes such as photosynthesis at the relevant spatial and temporal time scales.

Fig. 1: (left) 3D structure factors of Photosystem II generated by indexing and merging tens of thousands of single-shot diffraction patterns collected at LCLS. [from Kupitz 2014 with permission] (center) Structure of Photosystem II at 5Å resolution, from LCLS diffraction data [from Kupitz 2014 with permission]; (right) Detail of a single-shot diffraction pattern of Photosystem I showing fringes due to the sub-micron finite size of the crystal [Chapman 2011 with modifications].