Serial femtosecond X-ray crystallography (SFX) is a
rapidly evolving new method for structure determination using thousands of
microcrystals and femtosecond, highly brilliant X-ray pulses from a
free-electron Laser (FEL) [1]. A single diffraction pattern is recorded per
crystal in a diffraction-before-destruction approach rendering this method
virtually radiation-damage free. In the past 3 years a low-dose, serial method
of data collection has been adapted to synchrotron radiation use at highly
brilliant micro-focus beam lines, allowing room temperature data collection
with data quality sufficient for de novo phasing [2]. It has also been shown
that exposure times of less than 3ms were sufficient for collecting serial
room-temperature data using for example a lysozyme microcrystal suspension [3].
The ability to collect room temperature data opens up the possibility of time
resolved studies, as bio-macromolecules can now be probed in their native
environments.
Despite this new method of serial crystallography holding great promise for macromolecular biological studies, the major bottleneck to date is having a reliable method for determining the quality of the thousands of microcrystals. Particularly for FEL application, the crystals are often so small they do not yield determinable diffraction at a synchrotron, let alone at an in house X-ray source. To make matters worse, beam time is scarce, making it essential to reliably distinguish protein nanocrystals from amorphous precipitate and ideally even diffraction quality of the crystals prior to accessing the beam. However, measuring at room temperature and collecting a single diffraction pattern from thousands of identical crystals makes it possible to use this technique to study enzymatic reactions of proteins in substrate mixing and substrate caging approaches.
We will present various methods for reliably growing microcrystals for SFX application along with different methods for determining crystal quality in preparation for an FEL or synchrotron beam time. In addition, special attention will be paid to scoring and handling of the crystal slurries, essential for structure determination using serial crystallography techniques. And finally, preliminary results towards applying serial crystallography at synchrotrons towards time-resolved studies will be presented.