Ultrafast Biophysical Studies of Biomolecules at the APS

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

In recent years, we have focused most of our efforts at the APS on time-resolved SAXS/WAXS studies of biomolecules in solution using the pump-probe method. Briefly, a laser pulse (pump) photoactivates or thermally excites a biomolecule, after which a suitably delayed X-ray pulse (probe) passes through the sample and records a scattering pattern on a 2D detector. It is well known that the SAXS region of the scattering pattern reports on the size and shape of the biomolecule, while the WAXS region is sensitive to secondary and tertiary structure. Time-resolved SAXS/WAXS scattering patterns therefore provide 'fingerprints' that can be correlated with protein structure via molecular models and can help assess which models best describe reaction pathways in solution. However, the full potential of these measurements has not yet been realized as scattering in the WAXS region is much weaker than in the SAXS region, and has required the development of experimental methodologies that not only maximize the signal-to-noise ratio of these measurements, but also identify sources of systematic error and correct for them. Due to the SARS-CoV-2 pandemic, the Advanced Photon Source (APS) excluded external users for well over a year. During that hiatus and continuing to the present, we embarked on a major overhaul of the software developed by our group to operate hardware employed on the BioCARS beamline. In addition, we continue to develop improved methods for analyzing the x-ray scattering data in both SAXS and WAXS regions and properly correct for sources of systematic error. The LaueCollect package developed by Dr. Schotte for operating the BioCARS 14-IDB time-resolved x-ray beamline exceeds 100,000 lines of code, and as it continued to evolve, it became increasingly difficult to manage. Consequently, we sought to develop a more modular, hierarchical, and extensible framework for creating code that would take full advantage of our 3rd generation Field Programmable Gate Array (FPGA) electronic timing system and the player-piano paradigm we developed to operate the FPGA. We came up with a Methods-based approach whereby a user can define a Method whose associated configuration parameters fully specify the precise timing at which time-resolved x-ray diffraction images are to be acquired, as well as the order of their acquisition. Configuration parameters can specify a variety of dependent variables including time delay between laser and x-ray pulses, sample temperature, laser power, motor position, etc., and also specify their order of acquisition via nested loops. With this paradigm, each component that plays a role in data acquisition is controlled by a 'soft' IOC (input-output controller module) that receives instructions and replies via network communication according to the channel-access paradigm. With this approach, complex data acquisition sequences can be executed routinely by simply recalling a previously defined Method from a pull-down menu and launching the data acquisition sequence. Since the APS reopened to outside users in the summer of 2021, we have had several opportunities to test and refine various aspects of this new paradigm. Though more work remains to be done, we hope to finalize the organizational structure of our new software framework and commission this software package on the BioCARS beamline before the end of 2022. One of the remaining technical challenges in our SAXS/WAXS studies is to fully extract the structural information embedded in time- and temperature-dependent scattering curves, in particular from the WAXS region. In a recent review article entitled "Time-resolved X-ray scattering studies of proteins," which was published in Current Opinion in Structural Biology in 2021, we pointed out that scattering studies published thus far have not yet achieved this goal. This effort is complicated by the fact that buffer and the fused silica walls of the capillary-based sample cell accounts for most of the scattering intensity in the WAXS region and requires accurate subtraction to isolate the far weaker contribution from the biomolecule of interest. Even when properly subtracted, the remaining scattering signal arises from both the biomolecule and its hydration shell. As pointed out in last year's annual report, time- and temperature-resolved changes in the hydration shell scattering can generate difference signals larger than those that arise from conformational changes in the biomolecule. Our efforts to develop improved methods that allow us to isolate the biomolecule contribution to the scattering with high accuracy and free of systematic error is ongoing. It all starts with the detector. The $1.6 M Rayonix MX340-HS X-ray detector employed on the BioCARS beamline at the APS consists of a 4x4 mosaic of 960x960 CCD chips (after 2x2 binning), each of which is coupled to a corresponding 2.92:1 fiber optic taper. The front surface of the fiber tapers is in contact with a thin phosphor film that absorbs x-rays and emits visible photons, some of which are coupled into the hexagonal array of "light pipes" that deliver the photons to the CCD chip. The use of fiber tapers to relay light emitted from a phosphor onto a CCD array allowed fabrication of much larger area detectors than would otherwise be practical due to the prohibitive cost of correspondingly large area CCD chips. When tuning the x-ray energy to 14 keV and positioning the detector about 189 mm from the scattering source, we achieve spatial resolution down to 1 , which is desirable for both crystallography and SAXS/WAXS studies. From a technical standpoint, this detector seems to represents a good match for our needs. However, our efforts to quantitatively characterize its statistics and response uniformity has exposed several complications that arise when employing fiber tapers. For example, due to aliasing arising from shifting registration between the hexagonal light pipes and the rectilinear pixel array, there is substantial variation in the coupling efficiency across the pixel array. To correct for coupling efficiency variation, as well as image distortion arising from the tapered fiber bundles, Rayonix developed a proprietary method to seamlessly map raw pixels from each CCD in the 4x4 mosaic to a single virtual image (3840x3840). Their procedure produces a gap-free image on a rectilinear grid, but in a way that significantly alters the statistics, the origins of which need to be properly understood to properly analyze SAXS/WAXS scattering images. For example, our statistical analysis found significant variations in the CCD chip to CCD chip gain settings, as well as significant pixel-to-pixel gain variation within each chip. The resulting non uniformity across the 4x4 mosaic that makes up the virtual image distorts the WAXS scattering curves and needs to be corrected. Moreover, the virtual images generated by the detector are frequently contaminated by so-called "zingers", in which clusters of pixels randomly report signal levels well above the background, even up to saturation, and arise from radioactive decay or cosmic rays interacting with the phosphor, fiber taper, or CCD chip itself. By characterizing in detail the dark and light statistical variance for every pixel in the virtual image, we not only establish statistical criteria for identifying and rejecting zingers, but also for calculating statistically-weighted averages of measured scattering intensities, thereby providing the most accurate measurement possible with this detector. As our time-resolved SAXS/WAXS methodology becomes more precise and easier to use, we expect it to become an ever more important complement to time-resolved Laue studies and time-resolved optical spectroscopy studies of biomolecules, and will help provide a structural basis for understanding how biomolecules function.