Optimisation of Initial Hits
Initial hits fromsparse-matrix screens can range from microcrystals, needle clusters, thin plates and spherulites/sea urchins to well-formed single crystals ready for data collection. This from Terese Bergfors illustrates visual examples of each morphology alongside diagnostic classifications.
If your initial trial yields sub-optimal crystal forms, the next step is systematic optimisation. Interpreting your drops in the context of the macromolecular crystallisation phase diagram allows you to tailor precipitant and macromolecule concentrations precisely, shifting the system from excessive spontaneous nucleation into the metastable growth zone.
Phase Diagram Mechanics in Vapour Diffusion
In a standard vapour diffusion experiment, water diffuses from the mixed droplet into the more concentrated reservoir solution (mother liquor). As protein and precipitant concentrations rise, the drop enters the supersaturated labile zone, crossing the energy threshold required for spontaneous primary nucleation.
As nuclei assemble, soluble protein in the drop is depleted, driving the system back down into the metastable zone. Here, no new nuclei can form, and the remaining soluble protein is consumed exclusively by the growth of existing crystal lattices.
Observing your initial drop outcomes against this phase boundary guides your optimisation strategy:
- Amorphous precipitate / dense microcrystalline showers: The drop reached excessive supersaturation deep within the labile or precipitation zone.
- Solution: Reduce initial protein concentration, lower precipitant concentration, or shift temperature.
- Clear drops: The system remained undersaturated.
- Solution: Increase protein or precipitant concentration, adjust pH, or screen additive conditions.
- Needle clusters or spherulites: Nucleation occurred too rapidly along multiple axes.
- Solution: Employ fine-gradient grid screening or seed-based uncoupling.
Automated Grid Optimisation (Dragonfly & Mosquito)
The standard initial approach is to design a fine 96-well grid screen centred around your lead condition. Typically, this combines a horizontal gradient of precipitant concentration with a vertical pH gradient across your buffering range.
Custom 96-well optimisation screens can be prepared rapidly using the facility's Dragonfly liquid handler. Dispensing of the recipient drops is then performed on the Mosquito LCP robot.
Booking reminder: Both the Dragonfly and Mosquito must be booked in advance via PPMS prior to use. Instrument usage is covered by the plate ordering fee (current pricing schedules are available on PPMS).
Seeding Based Optimisation
If grid screening continues to produce disordered clusters, acicular needles, or high nucleation density, crystal seeding decouples nucleation from crystal growth, allowing single crystals to grow in milder conditions within the metastable zone.
The facility standard is Micro-seeding on the Mosquito:
- Harvest Crystal Material - Under a microscope, draw up crystal-bearing drops along with ~30 µL of matching reservoir solution using a micropipette. Transfer the suspension into a microcentrifuge vial containing a ceramic or PTFE Seed Bead (Hampton HR2-320). If crystals were grown at 4 °C or 16 °C, pre-chill the tube and buffer on ice.
- Mechanical Seed Pulverisation - Vortex the Seed Bead vial at maximum speed for 30 seconds to fragment the crystals into sub-microscopic lattice nuclei, then immediately incubate on ice for 30 seconds. Repeat this cycle 5 times (reduce to 2–3 cycles for very fragile needles).
- Serial Dilution Series - Excessive seed concentrations cause crystal showers. Systematically prepare a serial dilution series in pre-chilled reservoir solution: neat (undiluted stock), 1/10, 1/100, 1/1,000, and 1/10,000.
- Automated Mosquito Dispensing - Load protein, seed dilution series, and reservoir condition onto the Mosquito LCP using the dedicated micro-seeding program. The robot dispenses a standard volumetric ratio of 100 nL protein + 20 nL seed suspension + 80 nL reservoir solution per drop.
Practical Considerations & Facility Support
- Ionic Compatibility: Micro-seeding carries over ~10% (v/v) of the parent reservoir buffer into recipient drops. Avoid incompatible ionic pairs, such as introducing sulphate-rich seed stock into screens containing calcium salts, which precipitates insoluble calcium sulfate.
- Alternative Seeding Techniques: For low-throughput or challenging targets, manual streak-seeding using animal whiskers or acupuncture needles, as well as single-crystal macro-seeding, can also be performed in the facility.
- Project Consultation: To discuss bespoke optimisation grids, additive screening, or seeding protocols, please contact the facility manager, Dr Sofia Caria (s.caria@imperial.ac.uk).