Solving X-ray protein structures without a crystal: using X-ray Free Electron Laser, the fourth generation synchrotron light sources

Abstract

A synchrotron light source is a source of electromagnetic radiation artificially produced by specialized electron accelerators. Compared to the commonly used in-house X-ray sources, it is wavelength adjustable, much stronger and more focused. In the last two decades, synchrotron usage has become the mainstream for X-ray protein structure determination. Taking the advantage of micro-focus light beams of the third generation synchrotron, the size of a usable protein crystal for data collection decreases to micron level, which increases the rate of macromolecular structure determination to about 10 new protein data bank entries per day. The fourth generation synchrotron light sources not only inherit the above advantages of traditional synchrotron but also develop them to a higher level: you do not even need a protein crystal for atomic resolution X-ray structure determination by using the nano-focus beam line at the fourth generation synchrotron! This is obviously exciting news for those structural biologists working on membrane proteins and large macromolecular complexes which are notorious for crystallization. X-ray Free Electron Laser (XFEL), provided by the fourth generation synchrotron light sources, has femtosecond pulse length and both peak and average brightness largely exceeding the beam of 3rd generation sources. XFEL is a laser that shares the same optical properties as conventional lasers such as emitting a beam consisting of coherent electromagnetic radiation which can reach high power, but it uses a relativistic electron beam as the lasing medium, which moves freely through a magnetic structure. In other words, it is coherent in phase and has very high brightness. The world's first XFEL, called Linac Coherent Light Source (LCLS), was completed at Stanford's Linear Accelerator Center in 2009 (Hand, 2009). Theoretically, a beam emitted by this machine will have “a brightness that is 10 billion times greater than that of any existing X-ray source on earth,” reported by the press office at the Argonne National Laboratory. At an XFEL, a nano-focus beam line is available. Together with the incredible intensity, a crystal with 1 μm might be large enough to yield the necessary scattering data. Of course the radiation damage will cause serious problems here. So people need to change their strategy for collecting a complete data set from rotating one crystal during exposure while collecting and merging the images from maybe hundreds of different crystals. It is unlikely that a nano-crystal could survive from one shot of XFEL. The very short pulse of XFEL also provides a way out of the damage problem. Compared to the picosecond pulse of a conventional synchrotron, XFEL could provide femtosecond pulse length. A computer simulation showed that one can rapidly image a sample with such a short pulse before the atoms in the molecule may be destroyed (Neutze et al., 2004). The most revolutionary impact of an XFEL on structural biology is the collection of X-ray scattering data without crystals. The basic idea of processing X-ray scattering data of single molecule is developed from electron microscopy (EM) studies (Huldt et al., 2003). In EM research, the images are sorted and averaged by the cross correlation between them. Then the mutual orientation of the averaged images can be determined based upon the idea that any two projections through a three-dimensional object have at least one same line. It is very similar in X-ray studies that any two scattering images share at least one curve in common where the Ewald spheres intersect in reciprocal space. By sorting and averaging the X-ray scattering images collected from