Abstract
X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.
Highlights
The determination of the structure of macromolecules at the atomic scale must contend with the effect of radiation damage
X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage
Only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors
Summary
The determination of the structure of macromolecules at the atomic scale must contend with the effect of radiation damage. With repetition rates of 120 pulses per second at the Linac Coherent Light Source (LCLS) [9], and capable detectors with corresponding frame rates [10], a natural approach to data collection is by recording serial snapshots from a flowing liquid suspension of protein crystallites This method of serial femtosecond crystallography (SFX) was first carried out at the LCLS in December 2009, at a long wavelength of 6 A (2 keV photon energy) on photosystem I and lysozyme crystals [5,11,12]. At higher intensities (or doses), it is expected that atomic scattering factors of heavier elements will ‘bleach’ owing to excessive ionization, providing a convenient method for phasing [26,27] This high-dose regime has not been fully explored, and it appears that we are still far from experiencing ultimate dose limits, even with the continued development of X-ray sources.
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