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.

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.

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

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

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)[/caption]Your content goes here. Edit or remove this text inline or in the module Content settings. You can also style every aspect of this content in the module Design settings and even apply custom CSS to this text in the module Advanced settings.

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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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