Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron
September 23rd, 2020
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The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.
Overview
This protocol demonstrates the preparation of serial crystallography samples using a high viscous injector, Lipidico, at the Australian synchrotron. It allows for the characterization of protein crystals embedded in viscous materials, facilitating easier data collection.
Key Study Components
Area of Science
- Crystallography
- Biophysics
- Material Science
Background
- Characterization of protein crystals is essential for understanding their structure.
- Existing methods struggle with soft materials and high viscous environments.
- Innovative techniques can improve sample preparation and minimize waste.
- The use of glass syringes allows for direct mounting onto the injector.
Purpose of Study
- To provide a method for preparing protein crystals in viscous media.
- To enhance the usability of the Lipidico injector system.
- To reduce sample waste during the crystallography process.
Methods Used
- Centrifugation of crystal solution to form a soft pellet.
- Loading of crystal solution into a glass syringe.
- Application of pressure to remove bubbles from the syringe.
- Direct mounting of syringes onto the injector for data collection.
Main Results
- Successful preparation of samples with minimal waste.
- Effective removal of bubbles to ensure sample integrity.
- Streamlined process for using the high viscous injector.
- Demonstrated feasibility of characterizing soft materials.
Conclusions
- The protocol provides a reliable method for preparing crystallography samples.
- It enhances the capabilities of existing crystallography setups.
- This approach can lead to better characterization of challenging materials.
Frequently Asked Questions
What is the purpose of using a high viscous injector?
The high viscous injector allows for the characterization of protein crystals embedded in viscous materials, improving data collection.
How are bubbles removed from the syringe?
Bubbles are removed by applying gentle pressure to create a seal and then quickly releasing the pressure to burst any bubbles.
What materials are used in this protocol?
Glass syringes and a high viscous medium are primarily used for sample preparation.
What is the significance of minimizing sample waste?
Minimizing sample waste is crucial for efficient use of materials and for maximizing the amount of usable data collected.
Can this method be used for other soft materials?
Yes, this method is designed to characterize various soft materials that are difficult to analyze with traditional setups.
Executive Industry Relevance
This protocol enables biopharma R&D teams to characterize protein crystals in high-viscosity environments, supporting structural biology workflows where traditional methods fail. By facilitating serial crystallography at room temperature using the Lipidico injector, it enhances data quality for target de-risking and lead optimization. The method improves sample efficiency and reproducibility, directly impacting early discovery decision-making and portfolio prioritization.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables structural characterization of protein targets embedded in viscous matrices, supporting functional validation and mechanistic insight.
- Operational Value: Reduces sample waste through efficient syringe-based preparation and direct injector mounting, improving throughput in target validation campaigns.
Screening & Assay Development
- Scientific Value: Produces homogeneous crystal suspensions in silicone grease, enabling reliable diffraction data collection for hit confirmation.
- Operational Value: Standardizes sample loading via glass syringes and coupler system, ensuring consistent injector performance across screening runs.
Translational & Preclinical Research
- Scientific Value: Supports room-temperature data collection, preserving native protein states relevant to physiological conditions and translational modeling.
- Operational Value: Integrates with synchrotron beamlines for high-throughput structural analysis, accelerating preclinical candidate evaluation.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through lead identification, providing structural data that informs hit-to-lead progression and assay refinement.
- Discovery Biology: Supports hypothesis testing by enabling high-resolution visualization of protein-ligand interactions in viscous environments.
- Screening: Delivers quantitative diffraction outputs that help compare binding modes and affinity trends across compound series.
- Analytics: Provides electron density maps and flow rate metrics that inform structural confidence and sample quality thresholds.
- Translational Research: Connects to preclinical work by maintaining protein integrity during data collection, supporting relevant biological interpretation.
- Enterprise Reuse: Establishes a reusable platform for serial crystallography across multiple targets and projects, reducing redundant setup efforts.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target structure, reduction of mechanistic ambiguity in viscous formulations.
- Operational Value: Standardization, reproducibility, and scalability of sample preparation and data collection.
- Strategic Value: Better go/no-go decisions based on structural data, capital efficiency from reduced sample consumption, and lowered biological risk in lead optimization.
- Portfolio Impact: Risk-adjusted prioritization of targets with validated structural data, enabling informed advancement decisions.
Implementation Considerations
- Requires expertise in protein crystallization, viscous sample handling, and synchrotron-based data collection.
- Depends on access to high-viscosity injectors like Lipidico and compatible beamline infrastructure (e.g., MX2 at Australian Synchrotron).
- Necessitates cross-team standardization between crystallography, formulation, and data analysis units for consistent sample preparation.
- Involves adaptation considerations for varying sample viscosities, with a lower limit of 10 Pa.s to maintain injector stability.
- Includes practical limitations such as potential stream instability with certain sample properties and the need for flow rate optimization to prevent curling or expansion of the extruded stream.
Biopharma FAQs
Why does bubble removal matter for serial crystallography data quality?
Removing bubbles ensures sample integrity and prevents flow disruptions during injection, which is critical for maintaining consistent diffraction data collection. The protocol uses pressure sealing and release or centrifugal force to eliminate air gaps, directly supporting reliable measurements at the injector tip.
How does crystal concentration in silicone grease affect injector performance?
Crystal concentration must be balanced with viscosity to maintain stream stability; too high a concentration risks exceeding the 10 Pa.s viscosity limit, while too low reduces data yield. Adjustments are made by varying silicone grease volume or diluting the crystal pellet in buffer prior to mixing.
What role does flow rate optimization play in stream stability for viscous samples?
Flow rate must be tuned to prevent extrusion defects: low rates cause silicone grease expansion, high rates produce thin streams, and intermediate rates yield stable extrusion. The Lipidico device flow calculator and incremental adjustments help achieve a slow but stable stream for reliable data collection.
Why is sample viscosity constrained to above 10 Pa.s for the Lipidico injector?
The injector’s design requires a minimum viscosity of 10 Pa.s to maintain pressure-driven flow without backflow or leakage; lower viscosities compromise stream control and sample delivery. This threshold ensures the high viscous injector functions as intended during serial crystallography runs.
How does electrostatic stabilization improve data collection for charged viscous samples?
For highly charged samples, a polystyrene catcher introduces a weak electrostatic force that counteracts stream curling, improving extrusion stability. This adaptation enables consistent sample flow and successful data collection, as demonstrated in the lysozyme crystal experiment.
Chapters in this video
0:05
Introduction
0:33
High Viscous Medium Crystal Preparation
2:37
Crystal Concentration Determination
3:28
Injector Mounting and Control
4:14
Sample Syringe Mounting
5:01
Running the Injector
5:59
Results: Representative Serial Crystallography Measurement
6:50
Conclusion
