Crystallisation Suite

The Crystallisation Suite is located on the ImperiaSouth Kensington Campus in the basement of the Sir Ernst Chain Building (Room 022). The facility provides instrumentation, consumables, and expertise to support macromolecular crystallisation at subsidised prices for Imperiamembers.

The suite is stocked with empty and pre-filled crystallisation plates alongside consumables for crystallisation experiments. Available liquid-handing robotics include Mosquito LCP, Dragonfly, and Tecan systems to enable rapid setup of 96-well crystallisation trials. 

If you are new to the facility, please visit Getting Started guide or contact the Facility Manager Dr Sofia Caria.

Crystallisation screens

The facility maintains 21 commercially available sparse-matrix screens pre-dispensed into 96-well MRC plates. Screens are stored at 4°C and can be ordered via PPMS for collection the following day. For an inventory of the commercial sparse-matrix screen, visit the Crystallisation Screens page.

Crystallisation robot

High-throughput nanolitre dispensing is  performed an SPT Labtech Mosquito LCP robot. Setting up a 96-well plate is quick (~5 minutes per plate). Pre-programmed dispensing protocols support a variety of experimental setups, including:

  • Variable protein to reservoir ratios.
  • Multiple proteins or concentrations in a single plate.
  • Micro-seeding.
  • Small molecule co-crystallisation.  

A standard 1-drop protocol for a 96-well plate (100 nL protein per drop) requires less than 10 uL of purified protein sample. Unless initial data or homologous structures indicate otherwise, a starting protein concentration of 10 mg/mL is recommended. For initial screening, can be prepared to further estimate ideal protein concentration experimentally.  

Plate storage

Three temperature-controlled, vibration-free crystallisation incubators operating at 20 °C, 16 °C, and 4 °C house crystallisation plates. Shelf space is assigned to research groups. Please do not move other groups' plates or occupy unassigned space.  If additional storage is required, contact the facility manager.

Plates are retained for a maximum of 6 months before disposal.

Crystal Observation & Imaging

The suite provides two Leica microscopes for plate inspection.  One microscope is equipped with a high-resolution CCD camera linked to a dedicated PC for live-view monitoring and image capture. A UV microscope is also available to verify whether crystals are macromolecular or inorganic salt by detecting intrinsic tryptophan fluorescence. Please note of false negatives or false positives.

  • UV positive:
    • Salt crystal coated by protein precipitation
    • Aromatic chemicals and small molecules
  •  UV negative:
    • Lack of Tryptophan
    • Fluorescence quenching (heme containing proteins, compound added or presence of DNA/RNA)
    • High solvent content
    • Material used in the experiment (type of tray or thickness of sealing tape).

Crystallisation Hit Optimisation

Following initial hit identification, the Dragonfly liquid handler can be used to dispense fine-gradient optimisation screens around your lead condition. Typical grid strategies combine a horizontal gradient of precipitant concentration with a vertical pH gradient across the buffer limits. 

Further optimisation pathways include:

  • Systematic screening of protein concentration and protein-to-reservoir drop ratios.
  • Incorporation of additives, ligands, or cryoprotectants.
  • Temperature (assess crystallisation at different temperatures: 20 °C, 16 °C, and 4 °C).
  • Seed-based crystallisation.

The facility manager is happy to discuss optimisation strategies based on your previous plates.  

Crystal Seeding Techniques

Seeding uses pre-formed crystal fragments to bypass the high activation energy barrier of primary spontaneous nucleation in the labile phase. Introducing seeds uncouples nucleation from growth, allowing controlled single-crystal development within the metastable zone at lower protein and precipitant concentrations. 

The facility supports three primary seeding modalities:

  • Micro-seeding: Mechanical crushing of micro-crystals into sub-microscopic nuclei for automated dispensing. This is the method of choice for 96-well robotic screening. 
  • Streak-seeding: Manual transfer of micro-fragments across a series of drops using animal whiskers, synthetic filaments, or acupuncture needles. 
  • Macro-seeding: Controlled physical transfer of an individual single crystal (20–100 µm) into a pre-equilibrated recipient drop to increase size or exchange ligands.

Robotic Micro-seeding Workflow

Seed Stock Preparation: Harvest crystal-containing drops into 30 µL of matching reservoir solution and transfer to a Seed Bead tube (Hampton HR2-320). Pulverise using 5 cycles of 30-second vortexing followed by 30 seconds on ice.

Serial Dilution: Dilute the stock serially in reservoir solution (neat, 1/10, 1/20, 1/100, 1/1,000, 1/10,000) to find the concentration that yields single crystals rather than showers.

Automated Dispensing: The Mosquito aspirates protein, seed dilution, and reservoir solution sequentially, dispensing a standard volumetric ratio of 100 nL protein + 20 nL seed stock + 80 nL reservoir solution per drop.

Storage: Working stocks may be kept at your incubation temperature; long-term stocks should be flash-frozen in liquid nitrogen and stored at -80 °C.

Precipitant compatibility note: Because micro-seeding introduces ~10% (v/v) donor mother liquor into recipient screen drops, avoid incompatible ion pairs (e.g. mixing sulphate-containing seed stock into calcium-containing screens will precipitate insoluble calcium sulphate).

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