Ultrafast Biophysical Studies of Biomolecules at the APS

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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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. Multiple sources of systematic error that compromise the accuracy of scattering measurements in both SAXS and WAXS regions have been identified, and we continue to develop strategies to characterize and correct these errors. These efforts require access to the BioCARS time-resolved beamline at the Advanced Photon Source (APS) near Chicago. Due to the SARS-CoV-2 pandemic, the APS excluded external users from 2020 up until the middle of 2021. In May 2023, the APS shut down for a major upgrade of its synchrotron ring, termed APS-U, and plans to resume user operations in the second half of 2024. The APS-U will no longer support their hybrid mode of operation and will thereby lower by about a factor of four the flux available in a single x-ray pulse. To ensure we are positioned to make effective use of weaker x-ray pulses when the APS resumes operation, we have focused much of our attention over this past year on the development and refinement of x-ray infrastructure, both hardware and software. This is not a small project, as the LaueCollect software package developed by Dr. Schotte for operating the BioCARS 14-IDB time-resolved x-ray beamline grew to over 100,000 lines of code, and as it continued to evolve, it became increasingly difficult to manage. We are currently developing a modular, hierarchical, and extensible framework for controlling the ever expanding hardware used to pursue time-resolved x-ray studies of biomolecules. Our new code aims to 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. A Methods-based approach is being developed whereby a user can define a named Method whose associated configuration parameters are saved in Tables that fully specify the order of data acquisition via nested loops as well as other dependent variables such as the time delay between laser and x-ray pulses, sample temperature, laser power, motor position, etc. Each hardware component that plays a role in data acquisition is controlled by a 'soft' IOC (input-output controller software module) that receives instructions and replies via network communication according to the channel-access paradigm. The Methods based Table organization is hierarchical, with the top-level Table consisting of columns that specify the name of the Method, the order of data acquisition, and a column for each IOC. Cells in the Table contain string commands that specify how the IOC is to behave during data acquisition. IOCs can be configured to change a variable on demand and reply 'Ready' when finished, or deterministically change a variable according to a trajectory whose start time is precisely phased with the FPGA and executed according to NTP time. With this approach, complex data acquisition sequences can be executed routinely by simply recalling a previously-defined Method from a pull-down menu and clicking a button to launch the data acquisition sequence. This approach is highly extensible, as IOCs developed to control new equipment can be made accessible to the Methods based data acquisition framework through configuration parameters, and not require extensive modifications to existing code, as was the case in the past. We are concurrently working on a user manual that explains our new framework for controlling the beamline, which should prove a valuable resource for both BioCARS staff and users. In addition to developing software to operate the beamline, we are developing new and improved methods for analyzing x-ray scattering data in both SAXS and WAXS regions, with a particular focus on how to identify and properly correct for sources of systematic error. The amount of image data acquired during a week of x-ray beam time can exceed four terabytes, and our new approaches aim to facilitate rapid and accurate processing of those data. X-ray images are acquired on a $1.6 M Rayonix MX340-HS X-ray detector that 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 use of fiber tapers to relay light emitted from a phosphor screen to an array of CCDs allowed fabrication of much larger area detectors than would otherwise be practical due to the prohibitive cost of correspondingly large area CCD chips. 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, 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. Our data acquisition sequence typically involves the measurement of 'air', 'capillary', 'buffer', and 'biomolecule' scattering images, with 'biomolecule' scattering images typically recorded at a range of concentrations that differ by factors of 3. It is crucial to properly scale the datasets so subtraction successfully isolates the contribution from the biomolecule. We have found that the capillary scattering is sensitive to its contents, and its SAXS scattering signature can change due to x-ray induced leaching of cations into and out of the capillary. We are working on schemes to characterize this effect and correct for it so we can recover accurately the curvature of scattering near q = 0, which is crucial to defining I0 and Rg, arguably the two most important parameters extracted from SAXS measurements. 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.