Biology: Time-Resolved Crystallography

While standard static 3-D structures of macromolecules obtained by using X-ray diffraction technique provide important insights into their function, to fully elucidate details of how these molecules perform their function, one ideally needs to capture them in action. Time-resolved crystallography is a unique tool for achieving this goal because it provides direct, detailed and global structural information as molecules in the crystal undergo structural changes.

BioCARS scientists played essential role in the early development of all aspects of time-resolved crystallography, including advances in processing and analysis of time-resolved data. BioCARS is one of only several synchrotron beamlines worldwide with infrastructure for conducting time-resolved experiments.

Time-resolved pump-probe experiments at BioCARS utilize polychromatic, Laue X-ray diffraction technique. For comprehensive reviews of time-resolved crystallography at BioCARS we suggest the following:

• Laue technique and examples of applications
  Ren et al. (1999) Laue crystallography: coming of age. J Synchrotron Rad 6, 891–917.
• Infrastructure for time-resolved crystallography at BioCARS 14-ID beamline
  Graber et al. (2011) BioCARS: a synchrotron resource for time-resolved X-ray science. Journal of Synchrotron Radiation 18, 658–670.
• Time-resolved crystallography principles and practice at BioCARS and LCLS
  Šrajer, V., and Schmidt, M. (2017) Watching proteins function with time-resolved x-ray crystallography. Journal of Physics D: Applied Physics 50, 373001.

Technical Capabilities

Time-resolution

• 100ps resolution in hybrid and 24-bunch APS storage ring mode
• 200ns in 324-bunch APS mode

X-ray flux

• two in-line undulators (U23 and U27), optimized for 12 keV
• polychromatic beam, 5% bandwidth typically used for time-resolved crystallography
• 3×10¹⁰photons/100ps pulse in APS hybrid mode

X-ray beam size

• 20 (h) x 20 (v) µm², beam focused by primary and secondary Kirkpatrick-Baez mirror systems

Repetition rate

• <40Hz typical, up to 1KHz possible

Lasers

• ps laser system: Spectra Physics, Ti:Sapphire Spitfire Pro 5; 780nm; 2ps; 1kHz; 5mJ/pulse; TOPAS OPA; tunable range: 350nm-2µm; pulses typically stretched to 30ps
• ns laser: OPOTEK Opolette 355 II HE; 7ns pulse duration; 20Hz; 410-600nm: ~3-6mJ; 230-400nm:0.5-2mJ
• a number of CW diode lasers; ms exposures possible

Detector

• Rayonix MX340-HS (10-100 frames/sec)

Challenges

While time-resolved crystallographic studies of reversible reactions in naturally photosensitive proteins can be routinely done, BioCARS staff is developing methods and capabilities to meet more challenging experiments.

Reaction initiation beyond light

• Use E-field jump, diffusion, T-jump, pH jump as methods of reaction initiation
• Hekstra et al. (2016) Electric-field-stimulated protein mechanics. Nature 540, 400–405.

Irreversible reactions

• Require rapid sample exchange
• Develop serial Laue micro-crystallography to facilitate studies of irreversible reactions while minimizing sample consumption
• Meents et al. (2017) Pink-beam serial crystallography. Nature Communications 8, 1281
• Martin-Garcia et al. (2019) High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source. IUCrJ 6, 412–425