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.
- 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.
- Henning et al. (2024) BioCARS: Synchrotron facility for probing structural dynamics of biological macromolecules. Structural Dynamics 11, 014301.

Addressing Challenges in Time-resolved Crystallography

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

- We 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.
- Lee et al. (2025) Direct visualization of electric-field-stimulated ion conduction in a potassium channel. Cell 188, 77-88.e15.

Studies of irreversible reactions by serial time-resolved Laue crystallography

- Irreversible reactions require rapid sample exchange
- We have developed 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, 1
- Martin-Garcia et al. (2019) High-viscosity injector-based pink-beam serial crystallography of microcrystals at a synchrotron radiation source. IUCrJ 6, 412–425
- Wilamowski et al. (2022) Time-resolved β-lactam cleavage by L1 metallo-β-lactamase. Nat Commun 13, 7379

Technical Capabilities

Time-resolution

- 250ps resolution in 48-bunch APS storage ring mode
- 100ns in standard APS mode

X-ray flux

- two U21 in-line undulators, optimized for 12 keV
- polychromatic beam, 3-5% bandwidth ~5.8×10⁹ photons/250ps pulse in APS 48-bunch mode at 200mA
- ~5.8×10⁹photons/250ps pulse in APS hybrid mode

X-ray beam size

- 25 (h) x 15 (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 (UV/VIS): OPOTEK Opolette 355 HE; 7ns pulse duration; 20Hz; tunable range: 210-2200nm; max energy @ 300nm: 1mJ/pulse; max energy @ 450nm: 8mJ/pulse
- ns laser (NIR): OPOTEK Opolette 532 LD; 7ns pulse duration; 20Hz; tunable range: 650-2400nm; max energy @ 1400nm: 6mJ/pulse
- CW diode lasers at various wavelenths; ms exposures possible

Detector

- Rayonix MX340-HS (10-100 frames/sec)
- ePix10K (250 Hz)

Crystallography Contacts

Robert Henning

Research Beamline Scientist
(630) 252-0446
henning@cars.uchicago.edu

Insik Kim

Research Beamline Scientist
(630) 252-2376
insik@uchicago.edu

Vukica Srajer

Research Beamline Scientist
(630) 252-0455
v-srajer@uchicago.edu

Laue Diffraction Pattern

Laue diffraction pattern collected at 14 ID using a tetrameric hemoglobin crystal from Scapharca Inequivalvis (courtesy of W. E. Royer, University of Massachusetts Medical School)

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Serial Laue Crystallography – Chip Scanning

Meents et al. (2017)

The micro-patterned silicon chip is raster-scanned through the X-ray beam using the goniometer at 14-ID beamline. A stream of humidified helium gas flowing over the chip prevents the crystals from drying out. Closer look illustrates short path of the direct beam between the collimator tube and the capillary beamstop behind the. By flushing the remaining free beam path with helium, extremely low background scattering levels are achieved.

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Serial Laue Crystallography – LCP Injector

Martin-Garcia et al. (2019)

Schematic diagram of the injector-based serial Laue crystallography at 14-ID beamline. Lipidic cubic phase (LCP) injector is used for delivering micro-crystals. Jülich chopper selects the desired number of 100ps X-ray pulses for X-ray exposure for each crystal. A Rayonix detector MX340HS records crystal diffraction. On the right, LCP is shown as mounted on the 14-ID beamline.

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Sample cell for room-temperature X-ray diffraction of protein crystals under strong electric field pulses. Protein crystals are sandwiched between two glass capillaries filled with crystallization solution and containing metal wires that serve as electrodes The crystal is fixed by an electrically insulating glue to the bottom (ground) electrode and the high-voltage pulse is introduced from a top electrode through a liquid contact with the crystal This electrode system was integrated into BioCARS setup for time-resolved crystallography.

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