Motivation and Aims of the AXSIS project

The vast majority of the >100,000 macromolecular structures that have been solved and are deposited in the protein data bank were determined by the method of X-ray crystallography. The intense and tunable X-ray beams produced by synchrotron radiation have made this amazing advance in structural biology possible. However, this success belies large challenges with the technique, which stem from radiation damage that is inherent to exposing soft matter to ionizing radiation. With the advent of X-ray FELs the new method of serial femtosecond crystallography (SFX), developed by some of us [Chapman 2011], initiated a new era in X-ray crystallography. It uses femtosecond pulses from X-ray FELs to obtain structures based on diffraction from a stream of nanocrystals in their mother liquor at room temperature, thereby outrunning radiation damage induced atomic motion (“diffraction before destruction” principle). Diffraction from crystals as small as 100 nm in width has been successfully observed, which contain less than 1000 molecules and are smaller than the micro-domains of most macroscopic protein crystals. The biggest advantages of nanocrystals are that they are much better ordered and are much easier to grow. The growth of large crystals has been the major challenge for the determination of structures of difficult to crystallize proteins like membrane proteins for which less than 600 structures have been determined so far. An example is Photosystem (PS) I, which consist of 36 proteins and 381 cofcators, where it required 13 years of effort to obtain macroscopic crystals (500 micrometer in width) that diffracted to near atomic resolution [Jordan 2001] in a synchrotron beam.

The SFX technique has the potential to determine the dynamics of proteins, [Aquila 2012, Kupitz 2014] by time-resolved SFX , where reactions are induced by light in the crystals “on the fly” before they are hit by the X-ray pulse, therefore molecular movies may be determined showing biomolecules at work. However, a current limitation of the technique is the limited access due to the worldwide only two current hard X-ray FEL facilities and their high cost of construction and operation. Moreover, while few-fs X-ray pulses can outrun the secondary X-ray radiation damage processes, as the fs pulses are faster than the motion of the atoms induced by ionization and breaking of chemical bonds, the electronic structure of the atoms is strongly disturbed in less than 5 fs by the loss of inner-shell electrons (formation of "hollow atoms") and valence electrons [Son 2011, Hau-Riege 2012]. Furthermore, the Self-Amplified Spontaneous Emission (SASE) mode at current FELs produces X-ray pulses, where each pulse varies in its exact field waveform, i.e., it misses longitudinal coherence. That is, the spectral photon distribution varies in each pulse and does not follow a Gaussian distribution but shows a complex spectral distribution with hundreds of maxima. This maps into the diffraction pattern and has very severe consequences for the accuracy of structure factor determination. Currently, more than 50000 indexed patterns and redundancies of 300-1000 for each reflection are required for the determination of accurate structure factors using the Monte Carlo integration method [Kirian 2011, Boutet 2012, Redecke 2013]. Reconstruction of the Bragg peak profiles could reduce the number of patterns required, but due to the random SASE spectra, this is extremely challenging. An attosecond X-ray source producing pulses with a "clean" and reproducible spectrum, i.e., full longitudinal coherence, will increase accuracy and require much fewer patterns. The attosecond pulses will outrun any damage, including the electronic states. The necessary high peak fluence can be achieved by using the naturally broad bandwidth attosecond pulses, which vastly improve the peak integration as compared with monochromatic X-rays.

Scheme of the attosecond serial crystallography and spectroscopy set-up. X-ray emission is measured with a multi-crystal spectrometer in the von Halmos geometry [Alonso-Mori 2012]. Diffraction is measured with a custom 1-kHz CMOS pixel detector

In the AXSIS-project, we develop attosecond serial X-ray crystallography and spectroscopy to push X-ray crystallography to a new frontier and overcome limitations of current fs-FELs to fuse atomic-resolution structure determination with ultrafast optical and X-ray spectroscopic techniques and extend the “probe before destruction” concept to the measurement of charge states and electronic transfer through X-ray emission (fluorescence) and absorption (near edge) spectroscopies. Ideally, the attosecond X-ray source is based on coherent inverse Compton scattering (C-ICS). The outline for such a setup, that we are developing and building during the course of this project, is discussed in more detail here. The nanocrystals under study are supplied to the X-ray beam in a liquid jet at room temperature, as with FEL experiments. While the current two hard XFELS are limited to 120 images/second (LCLS) and 60 images/second (SACLA), one thousand diffraction patterns and photoemission spectra will be obtained per second with the attosecond pulses of the C-ICS source. We can make a molecular movie by adding an optical stream of excitation pulses precisely synchronized to and preceding the X-ray pulses and varying the delay between optical and X-ray pulses. This unique tool will finally fulfill the dream of observing chemical reactions and biological processes in real space and real time at the necessary time and length scales of atoms and molecules. By developing this technique based on a compact laboratory-scale X-ray source, we vastly extend the availability of attosecond serial crystallography and spectroscopy to the general science community.