The dream of watching a protein function in real time with near atomic resolution has been realized using time-resolved Laue crystallography. To further this capability, which we helped develop at the ESRF in Grenoble, France, we have launched a major effort to develop a picosecond time-resolved X-ray source at the Advanced Photon Source (APS) in Argonne, IL. In addition to our efforts to develop improved hardware for making these measurements, which is summarized in a separate report, we have also made further progress in developing software for analyzing these data. Though the effort to develop a stand-alone package capable of analyzing Laue data has proven to be much greater than envisioned at the outset, it represents a critical component in our research. This package, called TReX, continues to evolve, but now contains most of the features required to analyze time-resolved Laue diffraction data and generate molecular movies that unveil protein structure changes in real time. ? ? The first step in analyzing crystal diffraction data, whether acquired with monochromatic or polychromatic X-ray radiation, is the indexing of diffraction spots recorded on a two-dimensional detector. Robust auto-indexing algorithms have long existed for monochromatic diffraction images, but Laue diffraction images, which are generated with a polychromatic X-ray source, are not amenable to those methods. Consequently, the analysis of Laue diffraction data has generally proven to be a time-consuming, off-line process that has required a significant amount of face time in front of a computer. To address this problem, a robust zone-based algorithm for auto-indexing Laue diffraction images has been developed by Dr. Eric Henry. When the image center and distance between the sample and the detector are prescribed, spots on the detector plane can be mapped onto a locus of possible zone-vector directions, the consensus of which identifies zone-vectors suitable for determining the orientation of the crystal. Moreover, zone-vector consensus provides a criterion for optimizing the center of the detector, knowledge of which is crucial to accurately predict the Laue diffraction pattern.? ? Merging redundant Laue data requires several scaling steps, each of which is error prone. Thus, the quality of Laue data has traditionally been inferior to data acquired with monochromatic methods. We are currently working on a ratio method that eliminates the need to scale redundant observations, as scaling is automatically accomplished by taking the ratio of laser on and laser off observations. We anticipate that electron density maps computed according to merged ratios will provide greater structural detail than achieved in the past. ? ? Most X-ray beamlines operate 24/7. Because the setup requirements for time-resolved studies are significant and the time allocated to our studies is limited, we generally pursue our experimental studies around the clock for several days straight before turning the beamline over to the next user. To make best use of this precious beam time, it is invaluable to have rapid feedback regarding whether the experiment is working well or not. To that end, our TReX software is designed to analyze diffraction images as fast as they are acquired. By analyzing and visualizing our data in real time, we are able to make better decisions regarding how to use our remaining beam time. ? ? We continue to refine our time-resolved Laue crystallography methodology. In the past, crystals mounted in capillaries would often suffer slippage when exposed to intense laser pulses. The laser pulse generates a pump-induced shock wave that propagates through the crystal and can cause the crystal to move to a new position inside the capillary. Note that a semi-rigid object will generally have three points of contact with a surface. When the surface is smooth, that object can easily skate around when perturbed. Crystal slippage causes the spot positions to smear out and compromises significantly the quality of our diffraction data. To arrest crystal slippage, we create texture on the inner glass surface by depositing a transparent polymeric film on the inside of the capillary. When the solvent carrying the polymer evaporates rapidly, the polymer surface becomes irregular. When the crystal is deposited on this irregular surface, it tends to be far more stable, and improves dramatically the quality of our diffraction data. In addition to this innovation, we are developing more automated crystal alignment procedures that allow us to acquire data over longer stretches of time without intervention. The infrastructure assembled at the APS for pursuing these studies is state of the art, and the time required to acquire a complete data set is now down to a few hours.
| Aranda 4th, Roman; Levin, Elena J; Schotte, Friedrich et al. (2006) Time-dependent atomic coordinates for the dissociation of carbon monoxide from myoglobin. Acta Crystallogr D Biol Crystallogr 62:776-83 |
| Bourgeois, Dominique; Vallone, Beatrice; Arcovito, Alessandro et al. (2006) Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography. Proc Natl Acad Sci U S A 103:4924-9 |
