Time-resolved structural studies at synchrotrons and X-ray free electron lasers: opportunities and challenges

https://doi.org/10.1016/j.sbi.2012.08.006Get rights and content

X-ray free electron lasers (XFELs) are potentially revolutionary X-ray sources because of their very short pulse duration, extreme peak brilliance and high spatial coherence, features that distinguish them from today's synchrotron sources. We review recent time-resolved Laue diffraction and time-resolved wide angle X-ray scattering (WAXS) studies at synchrotron sources, and initial static studies at XFELs. XFELs have the potential to transform the field of time-resolved structural biology, yet many challenges arise in devising and adapting hardware, experimental design and data analysis strategies to exploit their unusual properties. Despite these challenges, we are confident that XFEL sources are poised to shed new light on ultrafast protein reaction dynamics.

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)

  • J. Hajdu

    The challenge offered by X-ray lasers

    Nature

    (2002)
  • K. Moffat

    Time-resolved macromolecular crystallography

    Annu Rev Biophys Biophys Chem

    (1989)
  • K. Moffat

    Laue crystallography: time-resolved studies

  • R.T. Aranda et al.

    Time-dependent atomic coordinates for the dissociation of carbon monoxide from myoglobin

    Acta Crystallogr D Biol Crystallogr

    (2006)
  • V. Srajer et al.

    Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography

    Science

    (1996)
  • J.E. Knapp et al.

    Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin

    Proc Natl Acad Sci U S A

    (2006)
  • B. Perman et al.

    Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle

    Science

    (1998)
  • Z. Ren et al.

    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

    (2001)
  • H. Ihee et al.

    Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

    Proc Natl Acad Sci U S A

    (2005)
  • M. Schmidt et al.

    Five-dimensional crystallography

    Acta Crystallogr A

    (2010)
  • D.W.J. Cruickshank et al.

    Multiplicity distribution of reflections in Laue diffraction

    Acta Crystallogr A

    (1987)
  • D.W.J. Cruickshank et al.

    Angular distribution of reflections in Laue diffraction

    Acta Crystallogr A

    (1991)
  • S. Westenhoff et al.

    Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches

    Acta Crystallogr A

    (2010)
  • I. Schlichting et al.

    Crystal structure of photolysed carbonmonoxy-myoglobin

    Nature

    (1994)
  • K. Chu et al.

    Structure of a ligand-binding intermediate in wild-type carbonmonoxy myoglobin

    Nature

    (2000)
  • Z. Ren et al.

    Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures

    Proc Natl Acad Sci U S A

    (2012)
  • R.H. Baxter et al.

    Time-resolved crystallographic studies of light-induced structural changes in the photosynthetic reaction center

    Proc Natl Acad Sci U S A

    (2004)
  • A.B. Wohri et al.

    Light-induced structural changes in a photosynthetic reaction center caught by Laue diffraction

    Science

    (2010)
  • M.H. Stowell et al.

    Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron–proton transfer

    Science

    (1997)
  • A.B. Wohri et al.

    Lipidic sponge phase crystal structure of a photosynthetic reaction center reveals lipids on the protein surface

    Biochemistry

    (2009)
  • M. Cammarata et al.

    Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

    Nat Methods

    (2008)
  • R. Neutze et al.

    Visualizing photochemical dynamics in solution through picosecond X-ray scattering

    Phys Rev Lett

    (2001)
  • A. Plech et al.

    Visualizing chemical reactions in solution by picosecond X-ray diffraction

    Phys Rev Lett

    (2004)
  • J. Davidsson et al.

    Structural determination of a transient isomer of CH2I2 by picosecond X-ray diffraction

    Phys Rev Lett

    (2005)
  • H. Ihee et al.

    Ultrafast X-ray diffraction of transient molecular structures in solution

    Science

    (2005)
  • S. Subramaniam et al.

    Molecular mechanism of vectorial proton translocation by bacteriorhodopsin

    Nature

    (2000)
  • R. Neutze et al.

    Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

    Biochim Biophys Acta

    (2002)
  • Cited by (137)

    • Current trends in membrane protein crystallography

      2022, Advances in Protein Molecular and Structural Biology Methods
    • Crystalizing the interface – The first X-Ray structure of an oil/surfactant/brine transition layer

      2020, Journal of Petroleum Science and Engineering
      Citation Excerpt :

      Seeing is believing; in almost all scientific disciplines that involve chemistry there is a desire to go beyond the physical limits that prevent the observation of real-time and real-space motion of molecules (Zewail, 2000). In, for example, ultrafast sciences this has almost been accomplished, at least by inference, with the advent of pulsed and very bright X-ray sources operating in the femtosecond time domain (Neutze and Moffat, 2012), which can, in principle, produce snapshots of chemical reactions while they occur. However, the experimental conditions are very specific and in reality it is only gaseous unimolecular decomposition reactions that can be interrogated.

    • Homogeneous batch micro-crystallization of proteins from ammonium sulfate

      2021, Acta Crystallographica Section D: Structural Biology
    View all citing articles on Scopus
    View full text