Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography

Understanding the dynamic processes of life requires detailed knowledge of protein structural changes during function. Time-resolved serial crystallography (TR-SX) provides unprecedented insights into out-of-equilibrium conformations and reaction intermediates. A reaction is initiated in a protein crystal, followed by X-ray exposure after a defined delay. This process is repeated for thousands of crystals to obtain a 3D structure at that time point. Multiple snapshots create a ‘movie’ of the process. However, TR-SX requires specialized expertise and is often inaccessible to non-specialist groups. Many enzymes have moderate turnover kinetics, making them accessible via synchrotron radiation experiments. The authors aimed to bridge the gap between traditional and time-resolved methods by developing a readily accessible tool for time-resolved experiments, particularly focusing on biologically relevant timescales (sub-second). Previously developed methods such as HARE and LAMA offered in-situ mixing versatility, inspiring the development of the spitrobot to further facilitate time-resolved experiments and make them more accessible to a broader user base.

Traditional cryo-trapping methods for quenching enzymatic reactions in protein crystals suffer from limitations in manual substrate deposition and achieving accurate, reproducible delay times, particularly for fast timescales. Several studies have addressed this challenge. Warkentin et al. (2006) introduced hyper-quenching for protein cryocrystallography. More recently, Clinger et al. (2021) reported a 'mix-and-quench' device, but it relied on non-standard sample holders, limiting its adaptability. This paper builds upon these advancements by leveraging the advantages of existing high-throughput infrastructure.

The spitrobot comprises a plunger, humidity flow device (HFD), liquid application method (LAMA) droplet injector, vitrification chamber, camera system, and control unit. Crystals mounted on SPINE-standard MicroMesh™ sample holders are positioned on an electropneumatic piston within a humidity and temperature-controlled environment. The LAMA method initiates the reaction by delivering picoliter-sized droplets onto the crystals. After a defined delay, the crystals are plunged into liquid nitrogen. High-resolution cameras automatically capture images before and after droplet deposition. The SPINE standard simplifies integration into existing high-throughput workflows. The plunger uses an electropneumatic piston regulated by gas pressure (3-6 bar) for plunging speeds comparable to existing solutions. The HFD maintains humidity (4-40°C, up to 99% humidity) and temperature, allowing for controlled dehydration. The LAMA injector deposits picoliter droplets precisely onto the mesh using piezo-actuators and precise nozzle alignment assisted by a camera system. The vitrification chamber uses a standard foam dewar with an aluminum mount for SPINE-standard pucks. Dry nitrogen gas displaces gaseous nitrogen to improve vitrification rates, and the lid is heated to reduce ice formation. The vitrification time was characterized optically and electronically, using thermocouples of different sizes. The smaller thermocouple (13 µm), comparable to crystal size, minimized the Leidenfrost effect and showed a total processing time of ~50 ms, including air-valve delay, plunge time, and vitrification. The glass-transition temperature was reached within 7.5 ms. The camera system enables automated image acquisition, confirming droplet deposition and aiding in data collection. The methodology includes both serial synchrotron crystallography (cryo-SSX) and standard rotation data collection. For cryo-SSX, microcrystal slurries are loaded, excess mother liquor removed, and ligand solutions applied via LAMA. For single-crystal data collection, crystals are manually mounted, and the LAMA method initiates the reaction. The study includes xylose isomerase (XI), CTX-M-14 β-lactamase, and tryptophan synthase (TS) as model systems. Cryo-protection was achieved using 2,3-butanediol or ethanol. Data collection and processing were performed using standard crystallography techniques (CrystFEL, XDS, AutoPROC, PHASER, etc.).

The spitrobot successfully trapped reaction intermediates with millisecond time resolution. Ligand binding (glucose and 2,3-butanediol in XI; avibactam and ampicillin in CTX-M-14) was observed at 50 ms and 1 s, respectively, using cryo-SSX. Single-crystal rotation data confirmed covalent complex formation in CTX-M-14 E166A mutant with ampicillin at 0.5, 1, and 5 s. Glucose binding in XI was consistently observed at 50 ms, 250 ms, 500 ms, and 1000 ms, demonstrating access to biologically relevant timescales. Cryo-trapping of reaction intermediates in macroscopic TS crystals showed the formation of an external aldimine intermediate at 20 and 30 s, providing insights into the TS turnover reaction. The minimum achievable delay time was approximately 50 ms. The spitrobot's compatibility with SPINE standards streamlines integration into existing high-throughput beamlines. The results demonstrate that the spitrobot is a versatile tool suitable for a variety of crystal sizes, data collection methods, time-delays, and environmental conditions.

The spitrobot offers significant advantages over existing methods for time-resolved crystallography. Unlike most cryo-EM plungers, it incorporates reaction initiation capabilities. Compared to manual cryo-trapping, it achieves higher reproducibility and accuracy, particularly at fast timescales. It enables remote experiments, the testing of a few crystals before larger-scale experiments, and the use of crystals with unfavorable size-to-diffraction ratios. The capability to maintain physiological conditions during reaction initiation is a significant advantage. The study addresses limitations of manual procedures by providing precise time control and consistent results across time-scales and crystal types. While cryo-trapping introduces potential biases (e.g., side-chain conformations), the advantages outweigh the limitations when considering the wider accessibility and versatility of the method. The SPINE standard compatibility is a crucial aspect, facilitating widespread adoption and integration into existing infrastructure.

The spitrobot is a versatile and user-friendly tool for time-resolved crystallography, enabling millisecond cryo-trapping with high reproducibility and compatibility with standard high-throughput workflows. Its accessibility and versatility make it suitable for a wide range of applications and user groups, pushing the boundaries of our understanding of dynamic biological processes. Future work could focus on further optimizing the system's speed and automation and broadening the range of applicable biological systems.

The minimum achievable delay time of 50 ms might limit the study of ultrafast processes. Cryo-trapping inherently introduces potential biases, requiring careful consideration of the limitations of cryo-preservation on structural interpretation. The manual blotting of excess mother liquor may introduce variability, though image acquisition offers quality control. Crystal size homogeneity is crucial to avoid biases due to varying diffusion times.