Since its birth in the 1960s, biocrystallography has been a primary source of structural information, contributing more than 90% of 3D structures accessible in the Protein Data Bank [1
] and remains a central player in structural biology, alongside NMR and CryoEM. Over the past decade, new experimental setups have been introduced that widen its applicability and transform the daily practice of crystal growers and crystallographers. The recent advent of X-ray free electron lasers (X-FEL) has enabled the serial femtosecond diffraction (SFX) analysis of micro- and nano-crystals, and offers unprecedented possibilities for time-resolved experiments [2
]. At the same time, electron microscopes have been hijacked to perform micro-electron diffraction (µED), opening the way to the characterization of nanocrystals using laboratory-based instruments [5
]. Though, like conventional ones relying on synchrotron or neutron sources, these new crystallographic approaches require crystalline material and call for the development of means facilitating the production of calibrated samples (i.e. nano-, micro-, or macrocrystals) with a size adapted to the radiation (electrons, X-rays, or neutrons) and the experimental setup.
Growing crystals of a new biomolecule (protein, DNA, RNA, and their complexes) is often a time-consuming task that involves a trial-and-error screening step to find solvent conditions generating promising crystalline or microcrystalline phases. It is followed by an optimization step to improve the quality of one or several crystalline forms and make them suitable for diffraction analysis [9
]. However, before the first diffraction test, the evaluation of this two-step process mainly relies on optical microscopy observations. As a consequence, early crystal growth events, including nucleation, nano-crystal, or nano-cluster formation that directly impact the final crystallization outcome, remain hidden to the crystal grower. For this reason there is a clear need for a system enabling the preparation and the optimization of crystals under well-defined and controlled conditions, and ensuring reproducible crystalline properties and quality. The concept of such a system emerged in the nineties in the frame of a European research consortium on crystal growth (European Bio-crystallogenesis Initiative, 1998–2000) and was developed in the context of the OptiCryst European consortium (2006–2010) [10
]. The current implementation called XtalController (or XC900; Xtal Concepts GmbH, Hamburg, Germany) is composed of a crystallization chamber for a single experiment with precise temperature and humidity control [11
]. The composition of an initial drop (volume 5–10 µl) of a solution of the target biomolecule can be modified by the injection of various solutions (such as water, buffer or crystallant) using two piezo injectors spraying 70 picoliter droplets (Figure 1
A,A’). The sample drop can also be concentrated by evaporation of volatile chemicals (generally water) and its composition (i.e., the concentrations of components) is continuously calculated from its weight recorded to ±1 µg by an ultra-sensitive balance. The instrument provides diagnostic means to track the drop content along the experiment; the early occurrence of association events leading to nucleation, as well as nanocrystals can be detected in real time by dynamic light scattering (DLS) and the growth of crystals by video microscopy as soon as they reach a size exceeding a few microns. The XtalController also provides means to navigate in the phase diagram, from an undersaturated solution to a supersaturated state leading to crystal growth or precipitation. Hence, a specific phase may be stabilized or the system may be driven in the phase diagram toward another phase by varying experimental conditions in real time using the piezo injectors.
For more than three decades DLS has proven to be instrumental to study nucleation, to perform quality control of biological samples, to predict the propensity of the latter to crystallize, and, more recently, to follow their behavior in crystallization assays [12
]. With the unique and innovative combination of piezo injectors to modify the experimental conditions and DLS to track in real time the effect of various physicochemical parameters (chemical composition, biomolecule concentration, temperature) on biomolecules in solution, the XtalController opens a wealth of possibilities for basic and applied crystallogenesis. First examples included the observation of liquid dense clusters formed during nucleation [17
] and the preparation of crystals with well-defined size [18
Here we used this technology to study the crystallization of two enzymes, the CCA-adding enzyme from the psychrophilic bacterium Planococcus halocryophilus
(PhaCCA) and the hen egg-white lysozyme (HEWL). In the first case, classical vapor-diffusion assays produced numerous small crystals or precipitates. The XtalController helped better define the appropriate crystallant concentration to nucleate and grow large crystals of PhaCCA. In the second case, we used the XtalController to highlight the nucleating effect of a lanthanide complex, the crystallophore Tb-Xo4 [19
] on HEWL in the absence of crystallant. Both examples illustrate the potential of this technology in crystallogenesis and for the design of protocols to produce calibrated crystalline samples for a variety of crystallographic applications.
2. Materials and Methods
2.1. Chemicals and Enzyme Samples
Chemicals used for the preparation of buffers and crystallization solutions were of highest purity grade. Solutions were filtered on 0.22 µm porosity Ultrafree-MC membranes (Millipore, Molsheim, France). PhaCCA (monomer of 420 amino acids, 48 kDa) was produced in Escherichia coli
cells, purified, concentrated to 5 mg/mL, and stored at 4 °C in 50 mM HEPES-NaOH pH 7.5, 100 mM NaCl as described previously [21
]. HEWL (monomer of 129 amino acids, 14 kDa) was purchased from Seikagaku Corp. (Tokyo, Japan, Cat. N° 100940), Roche (Basel, Switzerland, Cat. N° 10153516103) and Sigma-Aldrich (St. Louis, MO, USA, Fluka Cat. N° 62970-5G-F). It was used without any further treatment and dissolved in milliQ water (Roche) or in 10 mM sodium acetate pH 4.5, 40 mM NaCl (Seikagaku and Sigma) at concentrations ranging from 25 to 71 mg/mL. Lysozyme stock solutions were filtered prior to concentration measurement. The crystallophore Tb-Xo4 used as nucleant for HEWL was synthesized and purified as described [19
]. It was dissolved in water to prepare a 100 mM stock solution.
2.2. Crystallization in the XtalController
The humidity and temperature of the crystallization chamber of the instrument were set to 99.5% and 20.0 °C, respectively, one hour before starting an experiment to ensure the stability of experimental conditions. A drop of 10 µl of enzyme stock solution was deposited on a siliconized glass coverslip (Ø 22 mm) placed on the balance. One pump was loaded with the appropriate crystallant solution and the second with pure water. The crystallization chamber was closed and the protocol started with a 5 min step to monitor drop evaporation and compensate the loss of weight by injection of water to keep the drop weight constant. Several steps of crystallant addition, drop evaporation or dilution were scheduled to explore the phase diagram. The shooting frequency of the pumps was adjusted to vary the slope of concentration variations. DLS measurements and drop image capture were scheduled at regular time intervals (e.g., every 5 to 30 min) to follow nucleation, aggregation, or crystal growth events. At the end of the experiment, the coverslip was transferred onto a 24-well Linbro plate (HR3-110, Hampton Research, Aliso Viejo, CA, USA) for incubation.
2.3. Standard DLS Measurements
In parallel to DLS measurements performed inside the XtalController, a benchtop Nanostar light scattering instrument (Wyatt Technology, Inc., Santa Barbara, CA, USA) was used to record the effect of Tb-Xo4 on lysozyme. Total of 10 µl of Sigma lysozyme (71 mg/mL) were transferred into a quartz cell for DLS measurements at 20 °C. The drop was covered with 10 µl paraffin oil and the cuvette was sealed with Parafilm™ foil to prevent evaporation. Subsequently, 1 µl of a 100 mM Tb-Xo4 stock solution in 10 mM sodium acetate pH 4.5 was added. In control experiments the Tb-Xo4 solution was replaced by buffer. Data were corrected for solvent viscosity and refractive index.
2.4. Crystal Analysis
Crystals of PhaCCA were analyzed in cryogenic conditions at FIP/BM30A beamline (ESRF, Grenoble, France) using an ADSC Quantum 315r detector. A crystal was soaked for a few seconds in the mother liquor supplemented with 20% (w/v) glycerol, mounted in a cryoloop and flash-frozen in liquid nitrogen. 240 images were collected with a rotation of 0.5° and an exposure time of 1 s per frame, yielding a dataset at a resolution of 2.28 Å. Crystals of HEWL were analyzed at ambient temperature using a Rigaku FR-X diffractometer at the FRISBI platform (IGBMC, Illkirch, France) with an EIGER R 4M detector (DECTRIS) with a 2θ offset of 10°. Several crystals grown with Roche, Sigma, and Seikagaku lysozymes were tested and diffracted to up to 1.5 Å resolution but showed rapid decay because of radiation damage at ambient temperature. The exposure time and rotation speed were adapted accordingly to collect a full dataset from a crystal of Seikagaku lysozyme plunged in viscous Parabar 10312 (Hampton Research), mounted in a cryoloop and protected from dehydration using the MicroRT room temperature kit from MiTeGen. 720 images were collected with a rotation of 0.25° and an exposure time of 2 s per frame. Data were processed with the XDS package [22
2.5. Structure Determination
The structures of PhaCCA and HEWL were refined in PHENIX [23
] using PDB entries 6QY6 and 6F2I (cleared of solvent molecules and ligands), respectively, as starting models for initial rigid body adjustment. Several rounds of refinement and manual inspection in COOT [24
] were performed until convergence of Rfree
(calculated using 5% of reflections). The model of PhaCCA was refined using one TLS group and includes 365 residues (the N-terminal expression tag and the flexible loop encompassing residues 83–94 are not visible), two phosphate ions, two acetate ions, and three glycerol molecules present in the mother liquor and the cryoprotection solution. The model of HEWL was refined using anisotropic atomic displacement parameters (ADPs) and includes the full enzyme sequence, one sodium, two chloride ions, a full Tb-Xo4 complex and two additional Tb3+
positions clearly identified in the anomalous density map but for which the ligand was not observed because of low occupancy.
In the perspective of emerging time-resolved studies of enzyme:substrate systems using SFX and XFEL facilities, it becomes increasingly important to gain more control over sample production, quality and reproducibility. These two examples of enzyme crystallization highlight the usefulness of the XtalController to monitor the evolution of crystallization assays and to act on the process. Beyond helping define and optimize crystallization conditions, the XtalController and its integrated DLS module may also be an ideal tool to:
Explore phase diagrams of biomolecules with a direct feedback on nucleation events;
Study the stability of biomolecules in solution with respect to various parameters such as temperature, pH, ligands, etc.;
Determine the optimum conditions for introducing a cryoprotectant;
Ensure the reproducibility of crystals in the context of structural biology investigations, rational drug design, and fragment-based screening.
Produce calibrated nanocrystals on demand (difficult to monitor and control otherwise) for diffraction analyses using X-ray free electron lasers and µED, or, conversely, to promote the selective growth of large crystals for neutron diffraction.
More generally, this type of versatile instruments provides a more rational approach to crystallization and a great alternative to extensive blind screening. We do believe that this technology has a bright future.