Capabilities

To enable beamtime at the oversubscribed LCLS to be used for scientific discovery rather than experiment optimization, the SDB resource is developing tools to prepare for biomedical experiments and to characterize samples before and during beam time. This ranges from understanding the sample quality and properties to the state of the sample during complex sample delivery or during a dynamic process being studied.

The existing capabilities of SLAC will be enhanced through these developments, providing new tools in sample preparation and characterization, as well as new diagnostics to understand the sample's state. Furthermore, capabilities at SSRL (Stanford Synchrotron Radiation Lightsource) will be leveraged and augmented to use the synchrotron X-ray beam as a key tool to prepare for LCLS beamtime and for new science. Finally, spectroscopic capabilities for sample characterization at LCLS will be expanded.

The developments will make use of state-of-the-art XFEL capabilities and complement them with offline capabilities that represent innovative advances. These capabilities will increase the impact of combined technologies on biomedical research by providing higher readiness levels to LCLS experiments for higher quality data in a shorter time. By increasing efficiency, the “extra” beam time access is used for additional higher-risk, higher-reward experiments, allowing faster access to emerging scientific questions.

By taking XFEL sample preparation and delivery from an art to a quantitative science, it is expected that LCLS and the developments of the SDB resource will have a significant impact on the structure determination of complexes and membrane proteins, provide accurate active site structures of metalloenzymes, and provide enhanced and widely available capabilities to observe macromolecular dynamics using the combined powers of LCLS and SSRL.

Overview of the capabilities planned for deployment:

• Sample preparation and delivery tools and methods for the laboratory
  • Injector and buffer characterization
  • Look-up tables of jetting conditions
• Synchrotron capabilities synergistic with LCLS
  • Deployment of same injector at LCLS and SSRL
  • Enhancement of the SSRL-LCLS interface with screening and phasing at SSRL
  • Deployment of rapid mixing injector and sample testing at SSRL including X-ray emission spectroscopy (XES)
• · Spectroscopic monitoring for metalloenzymes and time-resolved studies
  • Spectroscopic characterization in lab: UV-Vis
  • Vertical XES in all standard configurations
  • Integration of UV-Vis is standard configuration at LCLS starting with a goniometer
  • Deployment of usable laser system in lab for pre-characterization

High-intensity XFEL beams used to probe structures of a sample destroy the sample after exposure to a single X-ray pulse. As a result, experiments at LCLS require a sample delivery system that can replace the damaged sample between every X-ray pulse.

Crystal injectors were the first crystal delivery method used for serial femtosecond diffraction. They are commonly used at XFEL facilities because they can efficiently deliver many crystals, reduce background scattering, and enable new classes of time-resolved studies. Sample injectors produce a thin stream of crystals or sample solution by ejecting suspension through a small orifice. X-ray pulses at a high repetition rate interrogate the crystal stream and a diffraction pattern is produced each time a crystal and an X-ray pulse coincide.

While it is possible to collect serial data at cryogenic temperatures with rapid scanning fixed target systems, injectors can replenish room temperature samples at faster rates. Moreover, many biomedical problems require the unique features offered by sample injectors, including methods that use rapid mixing to study biomolecular dynamics.

The efforts of the SDB resource focus on advancing injector-based sample delivery methods to enable biomedical research, which includes solving new structures of membrane proteins and other radiation-sensitive biomolecules and enabling the study of protein dynamics such as metalloenzymes. These efforts will be achieved through the development of remote access capabilities for data collection for the Lipidic Cubic Phase (LCP) and other viscous media samples, as well as through the thorough testing and optimization of rapid mixing injectors.

These capabilities aim to maximize the unique LCLS beam's efficiency to promote greater access to the biomedical community and new scientific discoveries.

Overview of the capabilities planned for deployment:

• Modular and mail-in viscous media injectors that reduce sample consumption and enable remote access
  • Mail-in room temperature sample shipping
  • Mail-in screening
  • Rapid exchange of reservoirs
• Liquid mixing injectors for rapid diffusion of reactants for time-resolved studies for SFX
  • Development of mixing injector characterization and optimization
• Liquid mixing injectors for rapid diffusion of reactants for time-resolved studies for Small/Wide-Angle X-ray Scattering (SAXS/WAXS) and Fluctuation X-ray Scattering (FXS)
  • Development of mixing injector characterization and optimization

The automation of LCLS structural biology experiments, using injector-based sample delivery, streamlines the process of solving new structures and examines the dynamic processes of biomolecules using mixing injectors and photoexcitation to trigger reactions.

Significant effort is required to fully automate LCLS experiments. These efforts include adding new controls and feedback sensors, implementing advanced algorithms to monitor the experiments, and making corrections or flagging for intervention when appropriate. Effective macromolecular crystallography beams at synchrotron sources show how decades of innovation can help achieve these efforts./p>

The Stanford Synchrotron Radiation Lightsource (SSRL) Structural Molecular Biology (SMB) program is adept in experimental automation and was the first synchrotron beamline in the world to offer a remote-access program to users, establishing a new paradigm for access and structural genomics.

The SDB resource is following the successes of synchrotron light sources in using automation to achieve similar accomplishments at LCLS, including automating sample delivery, data collection, data analysis, and feedback for experiments at room temperature.

Shorter times to collect datasets with real-time feedback on data quality and completeness allows more structures to be determined, which in turn allows users who are measuring a dynamic process, such as an enzymatic reaction, to get a more complete structural time series. In the case of time-resolved experiments, feedback on different data metrics will also be collected. More effective and, consequently, shorter data collection times will allow more users access to the unique capabilities of LCLS, leading to a broader scientific impact in biomedical applications.

With the study of dynamics under near-physiological conditions being at the core of LCLS applications, these capabilities provide enhanced visible light excitation capabilities and injector alignment to support time-resolved studies, including the use of mixing injectors and laser illumination to trigger reactions.

Using laser-released caged compounds will drive dynamics studies of G-protein-coupled receptors (GPCRs) and RNA polymerase-II. Laser-induced temperature jump capabilities will drive the goal of understanding protein dynamics by exciting them out of equilibrium and tracking the time evolution of the system.

SDB resource developments aim to achieve the scientific goals of LCLS users by improving reliability and automation throughout the experimental process.

Overview of the capabilities planned for deployment:

• Automated sample delivery and data collection
  • Automation of sample delivery and data collection
  • Equipment protection
  • Monitorization and optimization of pre-characterized variables (sample delivery quantities and spectroscopic information)
• Photoexcitation and ultrafast time-resolved studies
  • Automation of photoexcited experiments
  • Integration of laser diagnostics
  • Caged compounds
  • THz illumination
  • T-jump
• Automated data analysis and feedback
  • Integration of automated analysis
  • Phasing methods
  • Development of automated SAXS/WAXS analysis