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

Time-resolved crystallography provides structural snapshots of proteins during catalysis by initiating a reaction in crystals and probing at defined delays. While recent advances in time-resolved serial crystallography (TR-SSX) enable access to non-equilibrium conformations and intermediates, such experiments require multidisciplinary expertise and specialized setups. Many enzymes exhibit moderate turnover rates (~10 s⁻¹), accessible at synchrotron sources, and most macromolecular structures are still determined at cryogenic temperatures with established high-throughput workflows. Traditional cryo-trapping can quench reactions to capture intermediates but suffers from manual substrate application and poor control over fast, accurate, and reproducible delay times. To bridge the gap between traditional cryo-crystallography and TR-SSX using broadly available infrastructure, the authors developed the spitrobot: a SPINE-standard compatible crystal plunger integrating humidity/temperature control and in situ liquid application (LAMA) to enable millisecond-to-seconds time-resolved cryo-trapping.

The study builds on decades of kinetic protein crystallography and time-resolved macromolecular crystallography methods, including room-temperature TR-SSX and in situ mixing approaches such as HARE and LAMA. Previous flash-cooling devices and cryo-EM plungers provided vitrification capabilities, but typically lacked integrated reaction initiation with precise timing. A recent mix-and-quench device enables millisecond cryo-trapping but relies on non-standard holders and film-based mixing that limit long delays and complicate handling. The authors position spitrobot as a SPINE-standard, user-friendly solution that unites controlled reaction initiation with cryo-trapping across millisecond-to-seconds delays, facilitating widespread adoption at synchrotron beamlines.

Hardware and control: The spitrobot consists of (a) an electropneumatic piston-driven plunger, (b) a Humidity Flow Device (HFD), (c) a LAMA droplet injector, (d) a vitrification chamber with a SPINE puck mount inside a foam dewar, (e) a two-camera imaging system for alignment and QC, and (f) a control unit/software. SPINE-standard micromeshes are mounted on an electromagnetic holder attached to the piston. Environmental control (HFD): Provides humidified airflow at 4–40 °C with up to 99% relative humidity and typical flow rates of 20–35 l/min. Humidity and temperature are independently regulated (Arduino-based control) using heating resistors and ultrasonic nebulizers; an external cooler can be attached via heat exchanger. Stability tests showed humidity maintained within <1% after equilibration and temperature stability at 4, 10, 20, 30, and 40 °C. Reaction initiation (LAMA): A piezo-driven nozzle ejects 75 or 150 pL droplets from 50 or 70 µm ID glass capillaries at ~2 m/s. The nozzle is positioned 1–2 mm from the mesh using rail-mounted stages and aligned by two orthogonal cameras. Droplets are delivered in high-frequency bursts (2–6 kHz); typically 100–500 droplets per sample (15–75 nL) depending on mesh area, concentrations, and viscosity. Vitrification chamber: A foam dewar holds liquid nitrogen and an aluminum mount for rotating a SPINE puck through its 10 vial positions, aligning each position to the plunge point. A PMMA lid with a piston opening reduces icing; gaseous nitrogen above the LN2 is displaced and siphoned; the lid is heated to reduce frost. The piston plunges the mesh directly into LN2 and releases it into the puck automatically. Timing characterization: Optical LED strobing (400 Hz) synchronized to plunger motion indicated a piston motion duration of ~22.5 ms. Electronic temperature measurements using a small (~13 µm) K-type thermocouple matching crystal dimensions established a total processing time ~50 ms (including valve delay, plunge, and vitrification). Independent cooling tests showed the glass-transition temperature (~160 K, < −140 °C) reached within ~7.5 ms with a 90–10% fall time of 7.5 ms, corresponding to a cooling rate of ~2.3 × 10⁶ K s⁻¹. Intrinsic device delays: air-valve ~20 ms; piston motion ~25 ms; vitrification ~7.5 ms. Minimal achievable experimental delay is ~50 ms. Cameras: Two high-resolution cameras at 90° provide alignment and automatic imaging immediately before and after droplet deposition to verify deposition and spread, aiding subsequent data collection area selection. Sample preparation and data collection: - Cryo-SSX: Microcrystal slurry (~0.5 µL) is pipetted onto 700/25 µm micromeshes, excess mother liquor rapidly blotted, and ligand applied by LAMA (250–500 droplets, 2–5 kHz). After set delays (e.g., 50 ms for XI, 1 s for CTX-M-14), samples are plunged directly into SPINE pucks. Data collected as still images via mesh scans (beam ~7×3 µm FWHM, 12.7 keV, ~2×10¹² ph/s, 7.5 ms exposure; Eiger2 CdTe 16M). Processing with CrystFEL (XGANDALF) and molecular replacement in PHASER. - Single-crystal rotation: Individual microcrystals are centered using beamline mesh scans/heat maps. Rotation datasets collected with microfocus beams (~7×3 µm FWHM, 12.7 keV, ~4×10¹² ph/s, 7.5 ms frames) on Eiger detectors (P13/P14). Processing with XDS/autoPROC/STARANISO/CCP4 AIMLESS; MR in PHASER. POLDER/omit maps generated in Phenix; model building in Coot. Crystallization and reaction buffers: CTX-M-14 microcrystals (~20 µm) by batch in PEG8000/Li2SO4/NaOAc pH 4.5 with seeding; E166A mutant similarly. XI macrocrystals by sitting drop (PEG3350/Li2SO4/HEPES); XI microcrystals (~10×15×15 µm) by concentrating protein and vacuum evaporation. TS (TrpAB) purified by Ni-NTA and SEC; crystals by hanging-drop in PEG300/Tris/CsCl. Reaction initiation used sterile-filtered, degassed solutions: CTX-M-14 avibactam (0.5 M avibactam, salts, 15% 2,3-butanediol) or ampicillin (1 M Na-ampicillin, salts, 15% 2,3-butanediol) at 2 kHz; XI glucose (1 M) or glucose + 15% 2,3-butanediol at 5 kHz; TS mixture (17% PEG300, 0.1 M Tris pH 7.5, 20 mM CsCl, 10 mM G3P, 110 mM indole, 100 mM serine, 30% ethanol) at 6 kHz. Cryoprotection embedded in these solutions (e.g., 15% 2,3-butanediol or 30% ethanol for TS).

- Device performance: The spitrobot enables sub-second cryo-trapping with a minimal practical delay of ~50 ms. Intrinsic delays: air valve ~20 ms; piston motion ~25 ms; vitrification ~7.5 ms. Cooling rate ~2.3 × 10⁶ K s⁻¹. Plunger velocities ~1.6 m s⁻¹ at 3–6 bar. HFD maintains 4–40 °C and up to 99% RH with stable control. - Imaging and alignment: Automated high-magnification images before/after droplet deposition allow verification and guide data collection. - Cryo-SSX demonstrations: Xylose isomerase (XI) microcrystals showed 2,3-butanediol occupancy in the active site at 50 ms; occupancy persisted and was not replaced by glucose within 500 ms. CTX-M-14 microcrystals formed the avibactam complex at 1 s, with structures matching prior room-temperature SFX data (minor differences), confirming efficient in situ mixing and diffusion. - Single-crystal rotation results: CTX-M-14 E166A mutant formed covalent acyl-enzyme intermediates with ampicillin at 0.5 s, 1 s, and 5 s (consistent with prior work, e.g., PDB 7K2Y). XI:glucose complexes were successfully captured at 50, 250, 500, and 1000 ms, with clear difference density at each time point. - Macroscopic crystal intermediates: In tryptophan synthase (TS), external aldimine (Aex-Ser) intermediates in TrpB were visualized at 20 s and 30 s after mixing, with serine binding evident at 25 s and associated conformational changes in Lys87 and Gln114. These time points likely report on sequential cycles of the β-subunit reaction. - Compatibility and throughput: Direct plunge of SPINE-standard holders into pucks integrates with high-throughput synchrotron workflows and supports remote/automated data collection.

The spitrobot addresses a key bottleneck in time-resolved crystallography by combining precise in situ reaction initiation (LAMA) with controlled, rapid cryo-trapping on standardized SPINE hardware. It provides reliable access to biologically relevant millisecond-to-seconds delays that are hard or impossible to achieve manually, while maintaining crystals under near-physiological humidity and temperature until plunge. Demonstrations across micro- and macrocrystals show that ligand binding, covalent intermediate formation, and conformational transitions can be captured reproducibly at defined delays. The results align with previously established room-temperature TR-SSX findings (e.g., CTX-M-14:avibactam), supporting the validity of mixing and diffusion under the spitrobot workflow. Compared to other mix-and-quench approaches, the SPINE compatibility simplifies handling, enables long and short delays, and integrates seamlessly with beamline automation. Advantages include reduced radiation damage at cryogenic temperatures, improved reproducibility, and applicability at modest-brightness beamlines or home sources by decoupling the time-resolution from X-ray pulse timing. Care must be taken to control crystal size distribution and isomorphism to avoid diffusion time disparities or freezing-induced non-isomorphism when making mechanistic inferences.

This work introduces the spitrobot, a SPINE-standard compatible crystal plunger that unites environmental control, precise droplet-based reaction initiation, and rapid plunge vitrification to enable millisecond-to-seconds cryo-trapping for time-resolved crystallography. The device achieves a practical minimal delay of ~50 ms, vitrification within ~7.5 ms, and demonstrates successful capture of ligand binding and catalytic intermediates in multiple systems (XI, CTX-M-14, TS) using both serial and single-crystal rotation data collection. By simplifying workflows and ensuring compatibility with high-throughput infrastructure, the spitrobot lowers the barrier to time-resolved studies at synchrotrons. Future research can extend to broader enzyme classes, systematically explore diffusion versus turnover across crystal sizes, refine timing to push sub-50 ms limits, and integrate more automated image-guided targeting and data processing for fully unattended experiments.

- Minimal achievable delay is ~50 ms due to mechanical and cooling dead times, limiting access to faster events compared to some room-temperature TR methods. - Potential biases from cryo-trapping (e.g., altered side-chain conformations, hydration, freezing-induced non-isomorphism) can affect mechanistic interpretation. - Diffusion times depend on crystal dimensions; heterogeneous crystal sizes can introduce state heterogeneity and complicate isomorphous difference analyses. - Reliance on proper cryoprotection and humidity control; inadequate conditions may cause ice formation or lattice changes. - Non-mapped affiliation markers (not relevant to methodology) aside, the approach requires SPINE-standard hardware; non-standard mounts are not supported.