XFELs Open A New Era in Structural Chemical Biology

X-ray crystallography, the workhorse of structural biology, has been revolutionized by the advent of serial femtosecond crystallography using X-ray free electron lasers. Here, the fast pace and history of discoveries are discussed together with current challenges and the method's great potential to make new structural discoveries, such as the ability to generate molecular movies of biomolecules at work.

 Discovering the structure and mechanisms of the complete collection of biomolecules is one of the grand challenges of chemical biology. A new milestone was reached this year when the number of biomolecule structures solved by X-ray crystallography, NMR and electron microscopy exceeded 100,000. Although this number may seem to imply that structural biology is a mature field, we have only just scratched the surface of the structural diversity of biomolecules. Indeed, fewer than 600 membrane structures are known to date, of which fewer than 50 are human membrane proteins. Serial femtosecond crystallography (SFX) promises to extend the range of protein structures that can be solved by addressing several of the major shortcomings of X-ray crystallography. This Commentary discusses recent SFX advances and also points to challenges that must be resolved to make SFX available to the broad community in structural biology.

One of the major challenges in the structure determination of membrane proteins and large multiprotein complexes is the growth of crystals large enough and of high enough order for conventional X-ray crystallography. It can take years, if not decades, to solve the structures of these difficult-to-crystallize proteins. It would be ideal to determine structures from very small crystals, which can be much more readily obtained and also lack long-range disorder, but such nanocrystals are not suitable for conventional X-ray structure analysis because the dose limit of X-ray damage severely affects data collection of small crystals, even under cryogenic conditions at synchrotron sources.

Another major challenge of structural studies is capturing the dynamics of biomolecules. Most of the current X-ray structures provide only a static picture of the molecule. Time-resolved Laue crystallography, in which reversible photoinduced reactions are studied at synchrotron sources using a nonmonochromatic X-ray (a so-called 'pink beam'), has unraveled structural changes in proteins but requires very large crystals and so far has mainly been applied to light-driven reactions in small single-domain proteins1.

X-ray free electron lasers (XFELs), which provide short (femtosecond) X-ray pulses that are 1012stronger than that of a synchrotron, have begun to address these major challenges of X-ray crystallography. In SFX, data are collected in serial fashion, where crystals are delivered to the X-ray beam in their mother liquor at room temperature in a liquid jet (Fig. 1). The femtosecond X-ray pulses from an XFEL are so strong that they destroy any solid material, but the pulses are so short (1 fs = 10−15 s) that the X-ray diffraction snapshots are collected before the molecules and the crystals are destroyed. SFX thereby overcomes the size limitation of crystals and X-ray damage problem of conventional crystallography. The SFX data sets consist of tens of thousands of femtosecond X-ray diffraction snapshots, each collected from one single crystal in random orientation by intersection with a femtosecond X-ray pulse.

See Full Article Here