Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges
Highlights
► Recent advances in time-resolved Laue diffraction studies of light-sensitive proteins. ► Recent advances in time-resolved wide angle X-ray scattering (WAXS) studies of light-sensitive proteins. ► Proof-of-principle demonstrations of serial femtosecond crystallography using X-ray free electron laser radiation. ► Reflection on the challenges ahead for time-resolved diffraction and scattering studies at X-ray free electron lasers.
Introduction
Conformational dynamics are essential to the correct functioning of virtually all macromolecules expressed in the cell: all reactions involve atomic motion. Although X-ray crystallography has been applied with great success to solve the majority of known macromolecular structures, this method is static: it reveals a structure averaged in both time and space over the numerous conformations of a macromolecule within the crystal.
Two direct approaches to structural dynamics are offered by time-resolved Laue diffraction and time-resolved wide angle X-ray scattering (WAXS), which explicitly introduce the fourth dimension of time [1]. The current state-of-the-art relies upon brilliant, short X-ray pulses isolated by shutters and a rapid X-ray chopper [2]. Experiments are based on the pump-probe approach, in which a brief laser pulse (the pump) initiates a light-dependent structural reaction in the molecules in the sample, of which 10–40% are typically photoactivated; after a controlled, variable time delay, an X-ray pulse (the probe) interrogates their structure. The time delay is varied to cover the duration of the entire reaction, and (in Laue diffraction) the crystal orientation is varied to cover the unique volume in reciprocal space. Depending upon the chosen time delay and the complexity of the reaction mechanism, one or more intermediate conformational states may be sampled at each time delay. Fitting of the entire time course spanning all time delays enables several intermediates to be resolved, provided each attains a peak occupancy sufficient to be identified.
The time resolution of the pump-probe approach is generally limited by the longest of the laser pulse, the X-ray pulse and the jitter in the time delay. Both Laue diffraction and WAXS have achieved a temporal resolution of ∼100 ps [3, 4•], the duration of an individual X-ray pulse at synchrotron sources. In contrast, X-ray free electron lasers (XFELs) deliver extremely brilliant, highly coherent X-ray pulses of 10–100 fs in duration [5•], three to four orders of magnitude shorter than synchrotron-derived pulses. Lasing is based on a process known as self-amplified spontaneous emission (SASE) which very efficiently converts energy from the electron bunch, as it traverses a very long undulator, into the X-ray beam. The peak brilliance of the X-ray beam is approximately ten orders of magnitude greater than that attainable with any third generation synchrotron. This gain factor is the difference between taking a walk and traveling at the speed of light! [6] Unprecedented opportunities in X-ray science — including structural biology — are thus opened up; XFEL light sources offer a powerful example of disruptive technology. Indications of where these sources may lead us have begun to emerge through experiments conducted at the first XFEL to emit hard X-rays, the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. Here we review advances in time-resolved structural biology using synchrotron radiation and contrast them with approaches unique to XFEL sources.
Section snippets
Time-resolved Laue diffraction
Time-resolved Laue diffraction [7, 8] employs intense, polychromatic, synchrotron-derived X-ray pulses to collect Laue diffraction data from a single protein crystal after initiation of a light-dependent reaction by a short laser pulse. This ultimately yields a complete, high resolution set of time-dependent structure factor amplitudes from which the variation with time of the average conformational state of a protein is obtained. A time resolution of ∼100 ps to 5 ns was achieved in studies of
Time-resolved wide angle X-ray scattering
Time-resolved, synchrotron-based WAXS studies of proteins in solution [26] have recently emerged from similar studies of small molecules [27, 28, 29, 30] and provide an approach complementary to Laue crystallography. Time-resolved WAXS offers the key advantage that conformational dynamics are not constrained by intermolecular interactions present in a crystal lattice. This is more than a simple technical advantage since the reactions of many biological systems occur via relatively large
Coherent diffractive imaging at X-ray free electron lasers
Molecular dynamics simulations of a protein exposed to an exceptionally brilliant XFEL beam predicted that it would be possible to overcome the radiation dose limitations of traditional crystallography by rapidly collecting ultrafast X-ray scattering data before radiation damage had time to destroy a protein sample [43, 44]. Several experimental confirmations of this ‘diffract-and-destroy’ principle [45, 46, 47] were performed at the Hamburg VUV free electron laser FLASH [48]. In the hard X-ray
Serial femtosecond crystallography
SFX offers a radically new experimental approach to structural biology that, nevertheless, remains subject to the challenges of protein crystallization [63•]. SFX rapidly exposes individual, tiny liquid samples, each containing a randomly oriented nanocrystal or microcrystal, to a single, highly focused XFEL pulse. In a neat twist to the ‘diffract-and destroy’ concept, diffraction from crystals self-terminates as the protein molecules undergo an X-ray damage driven Coulomb explosion disordering
Conclusions: time-resolved structural studies with an XFEL source
Of the static structural approaches successfully demonstrated at an XFEL, SFX appears to be the most easily adapted to a time-resolved experiment. Indeed, initial time-resolved SFX studies of photoactivated microcrystals of photosystem I in complex with ferrodoxin [69•] showed that they suffered a loss in diffraction quality 10 μs after laser triggering, suggestive of a substantial structural change. This result illustrates both the potential of time-resolved SFX and possible shortcomings. On
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Hyotcherl Ihee, Marius Schmidt, Jasper van Thor and Linda Johansson for supplying figures and granting permission to use them. Research by RN is supported by the Swedish Research Council (VR) and the Swedish Strategic Science Foundation (SSF). Research by KM is supported by NIH grants P41GM103543 (formerly RR007707) and RO1GM036452.
References (75)
- et al.
Anisotropic picosecond X-ray solution scattering from photo-selectively aligned protein molecules
J Phys Chem Lett
(2011) - et al.
Direct observation of myoglobin structural dynamics from 100 picoseconds to 1 microsecond with picosecond X-ray solution scattering
Chem Commun
(2011) - et al.
Rapid readout detector captures protein time-resolved WAXS
Nat Methods
(2010) - et al.
Lipidic phase membrane protein serial femtosecond crystallography
Nat Methods
(2012) - et al.
Protein dynamics control the kinetics of initial electron transfer in photosynthesis
Science
(2007) - et al.
Ultrafast X-ray scattering: structural dynamics from diatomic to protein molecules
Int Rev Phys Chem
(2010) - et al.
Chopper system for time resolved experiments with synchrotron radiation
Rev Sci Instrum
(2009) - et al.
Watching a protein as it functions with 150-ps time-resolved X-ray crystallography
Science
(2003) - et al.
Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering
Proc Natl Acad Sci U S A
(2010) - et al.
First lasing and operation of an angstrom-wavelength free-electron laser
Nat Photon
(2010)
The challenge offered by X-ray lasers
Nature
Time-resolved macromolecular crystallography
Annu Rev Biophys Biophys Chem
Laue crystallography: time-resolved studies
Time-dependent atomic coordinates for the dissociation of carbon monoxide from myoglobin
Acta Crystallogr D Biol Crystallogr
Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography
Science
Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin
Proc Natl Acad Sci U S A
Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle
Science
A molecular movie at 1.8 Å resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds
Biochemistry
Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds
Proc Natl Acad Sci U S A
Five-dimensional crystallography
Acta Crystallogr A
Multiplicity distribution of reflections in Laue diffraction
Acta Crystallogr A
Angular distribution of reflections in Laue diffraction
Acta Crystallogr A
Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches
Acta Crystallogr A
Crystal structure of photolysed carbonmonoxy-myoglobin
Nature
Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin
Nature
Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures
Proc Natl Acad Sci U S A
Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center
Proc Natl Acad Sci U S A
Light-induced structural changes in a photosynthetic reaction center caught by Laue diffraction
Science
Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron–proton transfer
Science
Lipidic sponge phase crystal structure of a photosynthetic reaction center reveals lipids on the protein surface
Biochemistry
Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering
Nat Methods
Visualizing photochemical dynamics in solution through picosecond X-ray scattering
Phys Rev Lett
Visualizing chemical reactions in solution by picosecond X-ray diffraction
Phys Rev Lett
Structural determination of a transient isomer of CH2I2 by picosecond X-ray diffraction
Phys Rev Lett
Ultrafast X-ray diffraction of transient molecular structures in solution
Science
Molecular mechanism of vectorial proton translocation by bacteriorhodopsin
Nature
Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport
Biochim Biophys Acta
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