Abstract
The development and application of the free-electron X-ray laser (XFEL) to structure and dynamics in biology since its inception in 2009 are reviewed. The research opportunities which result from the ability to outrun most radiation-damage effects are outlined, and some grand challenges are suggested. By avoiding the need to cool samples to minimize damage, the XFEL has permitted atomic resolution imaging of molecular processes on the 100 fs timescale under near-physiological conditions and in the correct thermal bath in which molecular machines operate. Radiation damage, comparisons of XFEL and synchrotron work, single-particle diffraction, fast solution scattering, pump-probe studies on photosensitive proteins, mix-and-inject experiments, caged molecules, pH jump and other reaction-initiation methods, and the study of molecular machines are all discussed. Sample-delivery methods and data-analysis algorithms for the various modes, from serial femtosecond crystallo-graphy to fast solution scattering, fluctuation X-ray scattering, mixing jet experiments and single-particle diffraction, are also reviewed.
Highlights
The first hard X-ray free-electron laser (XFEL), the Linac Coherent Light Source (LCLS), began operation at the US Department of Energy SLAC laboratory near Stanford in 2009 (Pellegrini, 2012)
More importantly for structural biology, it has allowed time-resolved diffraction at room temperature, while avoiding most effects of radiation damage, in addition to allowing the study of submicrometre crystals that are too small for conventional crystallography
The ‘diffract--destroy’ method used outruns damage by using, for example, X-ray pulses of 40 fs in duration to produce an X-ray diffraction pattern before the onset of secondary damage from the growing cascade of photoelectrons, which subsequently destroys the sample. This has allowed ‘molecular movies’ to be made at atomic resolution using microcrystals at room temperature, without the need for cooling to avoid damage. This may be performed in the correct thermal bath in which these molecular machines operate, under near-physiological conditions. [While the volatile buffer present in crystals may differ from the working environment of a protein, many enzymes remain active in the crystalline form (Hajdu et al, 1988).] Since each sample is destroyed by a pulse, a continuously refreshed supply of hydrated
Summary
The history and invention of the most popular mode at present (SFX) can be traced to early proposals for the delivery of samples across a beam by liquid jet (Spence & Doak, 2004) and to the first applications and development of this method at a synchrotron (Shapiro et al, 2008) in preparation for its use in the first crystallography experiments at the LCLS (Chapman et al, 2011) using a gas-dynamic virtual nozzle (GDVN) for sample delivery (Weierstall et al, 2012) TR-SP can provide a true molecular movie without any form of ensemble averaging or modeling
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