Time-Resolved Crystallography

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Time-resolved crystallography: past, present and future › (PDF)

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:

Nanosecond time-resolved crystallography set-up at Sector 14, BioCARS, APS. Two shutters, synchronized with the RF clock of the APS storage ring, are used to isolate single X-ray pulses (100ps duration) or longer pulse trains: an ultra-fast rotating chopper is followed by a single-opening ms shutter. In pump-probe experiments structural changes in crystals are initiated by short laser pulses and probed by delayed X-ray pulses. Nd:YAG pumped dye or OPO lasers, with pulse duration of 4-7ns, are used routinely for photo-excitation of crystals. A ps laser system has been installed recently.

 

Important milestones for time-resolved research at BioCARS