The time-resolved set-up at Sector 14 was funded in part through a collaboration with Philip Anfinrud (NIH/NIDDK).
Studies of macromolecules by the X-ray diffraction technique over the last few decades have provided unprecedented insight into their function by providing the average, static 3-D structures of macromolecules. However, to fully elucidate how these molecules perform their function, one must watch them in action, along a reaction path that often involves short-lived intermediates. 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.
In time-resolved experiments, a reaction is rapidly triggered in molecules in the crystal, and short X-ray pulses are used to probe structural changes at various time delays following the start of the reaction. Time resolution of 100ps, matching the duration of a single X-ray pulse at the synchrotron source, has been achieved (Schotte et al., 2003). Reaction triggering is a crucial part of the experiment. The fastest method for triggering a reaction in the crystal involves use of ultra-short (fs to ns) laser pulses. This method is clearly suitable for inherently photosensitive molecules that undergo structural changes upon the absorption of light by an embedded chromophore. Alternatively, for proteins that are not inherently photosensitive, photo-triggering can be accomplished by using caged compounds.
Time-resolved experiments that require sub-second time resolution utilize the polychromatic, Laue X-ray diffraction technique, where the crystal is kept stationary during the X-ray exposure. A comprehensive review of the present, mature state of the Laue technique as well as examples of its application to static and time-resolved studies can be found in Ren et al., 1999. BioCARS staff scientists played an essential role in the development of all aspects of time-resolved crystallography. This technique has successfully advanced to a mature stage with the use of high-flux third-generation synchrotron sources, demonstrated ability to detect small structural changes even at relatively low levels of reaction initiation of 15-40% (Srajer et al., 1996; Ihee et al. 2005; Rajagopal et al., 2005) and with significant advances in processing and analysis of time-resolved Laue crystallographic data (Srajer et al., 2001; Ren et al., 2001; Schmidt et al., 2003; Rajagopal et al., 2004). A particularly important development is the application of the Singular Value Decomposition (SVD) method to the analysis of time-resolved crystallographic data (Ihee et al. 2005, Schmidt et al., 2003; Rajagopal et al., 2004; Rajagopal et al., 2005).
Time-resolved experiment: Data collection and analysis (see full-size figure for details). For a stationary crystal, a series of Laue images is collected before the exposure to a laser pulse (t=0) and at a number of time delays following the laser pulse. A similar series of diffraction images is collected for a number of crystal orientations to obtain a complete Laue data set. From measured time-dependent structure factor amplitudes, time-dependent difference electron density maps “dark-light” are calculated. SVD and post-SVD analysis is applied to obtain structures of intermediates states. Data for photoactive yellow protein is used for this illustration (Rajagopal et al., 2005). For details of data collection and analysis see M. Schmidt, H. Ihee, R. Pahl and V. Srajer, "Protein-ligand interactions probed by time-resolved crystallography," in Protein-Ligand Interactions: Methods and Protocols (U. Nienhaus, eds.), Humana Press, Totawa, NJ, pp. 115-154 (2005) (abstract).
Time-Resolved Crystallography at BioCARS
Since the beginning of time-resolved user operation at BioCARS in the fall of 2000, we have typically dedicated about three weeks of beamtime on the 14-ID beamline during each APS run to time-resolved and Laue experiments. The hybrid mode, a special APS operation mode (offered for 8-10 days per APS run), was used for experiments that require sub-µs time resolution, while the standard operation mode was used for experiments that require µs or longer time resolution. With the upgraded ultra-fast X-ray chopper, we are now able to utilize the standard 24-bunch mode for all time-resolved experiments, spanning time resolution from 100ps to ms and longer. This approach provides significantly more beamtime for these challenging experiments. The new ps laser system and the upgraded 14-ID beamline (two undulators in collinear configuration, KB mirror system) allow us to extend the time-resolution from ns to 100ps and to obtain diffraction images of small crystals by utilizing single 100ps X-ray pulses.
The time-resolved technique and its implementation at the BioCARS facility are described in detail in:
M. Schmidt, H. Ihee, R. Pahl and V. Srajer
Protein-ligand interactions probed by time-resolved crystallography.
In Protein-Ligand Interactions: Methods and Applications (G. U. Nienhaus, ed.), Methods in Molecular Biology, vol. 305, Humana Press, Totawa, NJ, pp. 115-154 (2005)
Time-resolved crystallography setup at Sector 14, BioCARS, APS. (Click image at left for larger, labeled view.) Short laser pulses are used to initiate structural changes in protein crystals. Both ps and ns lasers are available for photo-excitation. Structural changes are probed by 100ps or longer X-ray pulses. Precise timing electronics permits tuning of the time delay between the laser initiation pulse and the X-ray probe pulse from 100ps to seconds and longer. A single 100ps X-ray pulse can be isolated in both hybrid and 24-bunch mode by two shutters synchronized with the RF clock of the APS storage ring: an ultra-fast rotating chopper, followed by a single-opening ms shutter.
Important milestones for time-resolved research at BioCARS
- December 1998 -- First Laue experiment conducted at the 14-ID beamline
- October 1999 -- BioCARS acquires second-generation fast rotating chopper capable of isolating the single X-ray pulse in the APS hybrid mode; APS hybrid mode operation starts
- January-July 2000 -- Commissioning of nanosecond time-resolved experiments at 14-ID
- September 2000 -- Laue and nanosecond time-resolved user operation starts
- October 2000 -- Undulator-A replaces Wiggler-A at 14-ID; construction of the laser laboratory completed
- May 2003 -- New, automated Laue data processing software available (Precognition by RenzResearch, Inc)
- December 2006 -- Ultra-fast X-ray chopper upgraded to permit isolation of a single 100ps X-ray pulse in a 24-bunch mode of the APS storage ring (153ns spacing between X-ray pulses)
- January 2007 -- New ps laser system installed
- September 2007 -- Major upgrade to the 14-ID beamline nears completion: the new configuration is designed to extend the time-resolved crystallography capability to 100ps time domain, while utilizing the standard 24 bunch-mode of the APS storage ring
- December 2007 -- Commissioning of 100ps time-resolved experiments at 14-ID; first 100ps time-resolved data sets collected
- March 2008 -- First 100ps time-resolved experiments with external users