Protein crystallography using synchrotron radiation sources has had tremendous impact on biology, having yielded the structures of thousands of proteins and given detailed insight into their working mechanisms. However, the technique is limited by the requirement for macroscopic crystals, which can be difficult to obtain, as well as by the often severe radia-tion damage caused in diffraction experiments, in particular when using tiny crystals. To slow radiation damage, data collection is typically performed at cryogenic temperatures.
With the advent of free-electron lasers (FELs), capable of delivering extremely intense femtosecond X-ray pulses, this situation appears remedied. Theoretical considerations had predicted that with sufficiently short pulses useful diffraction data can be collected before the onset of significant radiation damage that ultimately results in Coulomb explosion of the sample1, 2. This has been shown recently at the first hard X-ray FEL, the LCLS at Stanford. High resolution data collected of a stream of microcrystals of the model system lysozyme agree well with conventional data collected of a large macroscopic crystal3. With the demonstration that de-novo phasing is feasible4, serial femtosecond crystallography has been established as a useful tool for the analysis of tiny crystals3, 5, 6, and thus the large group of proteins that resist yielding macroscopic crystals such as membrane proteins6. In addition to ensure the required fast exchange of the microcrystals upon exposure, liquid jet delivery has the advantage of allowing data collection at room temperature6,7, 8. This now only allows for time-resolved measurements, including at the chemical time-scale of femtoseconds, but also samples the room temperature structural distributions and dynamics naturally present in cells and probed biochemically in solution. As demonstrated recently9, this is important since structural dynamics and thus the observed conformation(s) is often temperature dependent. Monitoring ambient temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation9. Recent results will be described.