Fixed Target Serial Data Collection at Diamond Light Source
February 26th, 2021
We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24.
Serial synchrotron crystallography, or SSX, is a relatively new and rapidly developing area of MX.It's very well suited to low dose and room temperature experiments, but perhaps most importantly, it's really well suited to following dynamics. This allows stop-motion movies of proteins in action to be made. At I24, multiple modes of SSX are available.
This protocol focuses on fixed-target SSX, which allows a wide range of serial experiments using limited amounts of sample. The silicon chips can be damaged when loading and cleaning them, if you're unfamiliar with them. Also, the chip alignment protocol is completely new to many crystallographers, and the data processing is slightly different to your standard rotation data.
To prepare a chip holder, cut two sheets of polyester foil into approximately 6-by-6 centimeter squares, and place the sheets over the two base plates. Using metal sealing rings, fix the sheets in place, then carefully pull on the excess foil to remove any creases. Next, select a silicon chip with appropriately sized apertures relative to the size of the crystals to be analyzed.
Glow discharge the chip for 25 seconds at 0.39 millibar, with a 15 milliampere current. Use tweezers to place the silicon chip on the chip loading stage, with the raised bars facing down. Then, using a pipette, apply 200 microliters of the microcrystal slurry to the flat side of the chip, spreading the slurry to cover all the city blocks of the chip.
If the chip is damaged, cover any holes with a small piece of polyester foil, to ensure that an even vacuum can be applied. Then apply a gentle vacuum to aspirate all of the excess liquid through the chip. Remove the chip from the chip loading stage, and carefully blot the underside of the chip with filter paper to remove excess liquid.
Place the loaded chip on the larger half of the chip holder between the guide marks, flat side down. Place the small half of the chip holder onto the chip to seal it. The two halves of the chip holder should snap into place.
Then, using hex bolts, fix the chip securely in place. To align the chip, hold the chip holder at a 30-degree angle while approaching the mount. Then use the kinematic mounts to place the loaded chip onto the XYZ stage at the beam line.
When the magnets make contact, allow the chip holder to rotate parallel to the flow, and click into place. Once the chip is placed, use the beamline on-axis viewing system and the chip-alignment graphical user interface to locate the top left fiducial of the chip. To center on fiducial 0 in X, Y, and Z, move in and out of focus to align Z, up and down to align Y, and left and right to align X.Then click Set Fiducial 0"After aligning fiducial 1 and 2 with the X-ray beam in the same manner, click Make co-ordinate system"to generate a co-ordinate matrix.
Then click Block Check"to move the XYZ stage to the first well of each city block. If the X-ray crosshair lines up with the apertures, the chip is aligned. In this graphical representation of spot finding results from DIALS an updating hit-rate plot can be observed.
If a hit is clicked, the corresponding diffraction image will be displayed in the DIALS image viewer. In this table, current indexing and integration rates that update in real time as the data is collected during the visit can be visualized. Visualization of the unit cell parameters can reveal polymorphs.
Two-dimensional plots of useful parameters can also be produced to reveal variations that arise due to loading or dehydration effects. Stereographic projections can reveal the presence or absence of preferred orientations that can be fed back into the loading protocol. For example, in this projection, the effects of overloading a chip with lysozyme crystals can be observed.
When loading the chip, pipette slowly and use your finger to rest the pipette above the chip. This prevents you from touching the chip and accidentally stabbing holes in the silicon. When processing data, make use of the automated pipelines available.
This will give you a good idea as to how your experiment is progressing, and if you need to vary something. This method can be applied to any protein system that can be crystallized in significant quantities. And the reaction can be triggered by light X-rays or rapid mixing.
Dynamic studies on a series of structures can provide insight into how proteins function.
Overview
This article provides a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24. It emphasizes the methodology for conducting experiments with limited sample amounts while ensuring chip integrity.
Key Study Components
Area of Science
- Structural Biology
- Crystallography
- Protein Dynamics
Background
- Serial synchrotron crystallography (SSX) is a developing area in macromolecular crystallography.
- It is suitable for low dose and room temperature experiments.
- SSX allows for dynamic studies of proteins, enabling visualization of their actions.
- Fixed-target SSX can utilize limited sample quantities effectively.
Purpose of Study
- To provide a detailed protocol for fixed-target SSX at Diamond beamline I24.
- To ensure proper chip preparation and alignment for optimal data collection.
- To highlight the differences in data processing compared to standard rotation data.
Methods Used
- Preparation of silicon chips using polyester foil and sealing techniques.
- Application of microcrystal slurry to the chips and vacuum aspiration.
- Chip alignment using a graphical user interface at the beamline.
- Real-time data visualization and processing using automated pipelines.
Main Results
- Successful alignment of chips leads to accurate data collection.
- Real-time monitoring of indexing and integration rates during experiments.
- Visualization of unit cell parameters reveals polymorphs and loading effects.
- Dynamic studies provide insights into protein functionality.
Conclusions
- The protocol enhances the efficiency of fixed-target SSX experiments.
- Proper chip handling and alignment are critical for successful data collection.
- Dynamic studies using SSX can significantly advance our understanding of protein behavior.
Frequently Asked Questions
Executive Industry Relevance
Fixed-target serial synchrotron crystallography enables structural biologists to conduct dynamic studies of proteins using limited sample quantities, supporting target validation through high-resolution structural insights. This method reduces sample consumption while maintaining data quality, which is critical for early-stage discovery where protein expression and purification are bottlenecks. By facilitating room-temperature and time-resolved experiments, it enhances mechanistic understanding of protein function, informing lead optimization and de-risking efforts in preclinical development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by capturing protein conformations in action, supporting functional target validation.
- Operational Value: Reduces dependency on large crystal volumes, allowing screening of challenging targets with limited expression yields.
- Predictive Confidence: Provides structural data that informs binding site characterization and mechanism of action, improving target selection decisions.
Screening & Assay Development
- Scientific Value: Generates quantitative electron density maps from microcrystals, enabling precise ligand-binding assessments.
- Operational Value: Standardizes sample mounting and alignment via chip-based systems, improving reproducibility across screening campaigns.
- Assay Readiness: Produces real-time hit-rate and indexing metrics that support go/no-go decisions during fragment or compound screening.
Translational & Preclinical Research
- Scientific Value: Reveals polymorphic forms and conformational changes that may impact drug binding or stability, informing formulation strategies.
- Operational Value: Captures time-resolved structural data that can correlate with functional assays, strengthening translational biomarker alignment.
- Risk Mitigation: Identifies loading-induced artifacts or dehydration effects early, reducing risk of misinterpretation in downstream optimization.
Pipeline & Workflow Integration
Fixed-target SSX integrates into the discovery workflow following target identification and assay development, providing structural insights that guide lead identification and preclinical validation. It supports iterative design-make-test-analyze cycles by delivering rapid structural feedback on compound binding and protein dynamics.
- Discovery Biology: Supports hypothesis testing by visualizing ligand-induced conformational changes and protein dynamics relevant to mechanism of action.
- Screening: Enables assay readiness through standardized chip preparation and alignment, ensuring consistent data quality across compound screens.
- Analytics: Delivers quantitative outputs including unit cell parameters, hit rates, and integration metrics that allow objective comparison of experimental conditions.
- Translational Research: Connects structural observations to functional outcomes by revealing dynamics that may influence bioavailability or target engagement.
- Enterprise Reuse: Establishes a reusable platform for structural screening across multiple targets, reducing revalidation efforts in multi-project environments.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by providing high-resolution, time-resolved structural data under near-physiological conditions.
- Operational Value: Enhances reproducibility and scalability through standardized chip handling, alignment protocols, and automated data processing pipelines.
- Strategic Value: Improves capital efficiency by minimizing sample consumption and enabling rapid iteration in structural optimization campaigns.
- Portfolio Impact: Supports risk-adjusted advancement decisions by reducing mechanistic ambiguity in target-ligand interactions.
Implementation Considerations
- Requires expertise in macromolecular crystallography and familiarity with synchrotron-based sample handling techniques.
- Depends on access to beamline infrastructure with kinematic mounting, on-axis viewing, and automated data pipelines such as DIALS.
- Necessitates cross-team standardization of chip preparation, vacuum aspiration, and alignment procedures to ensure data consistency.
- Involves adaptation considerations for different crystal sizes and space groups, requiring adjustment of aperture selection and spreading techniques.
- Includes practical limitations such as chip fragility during loading and cleaning, and sensitivity to dehydration effects that may alter crystal quality over time.
