Chapter 2. Materials and Methods
2.10. Protein crystallisation
2.10.1. Vapour diffusion crystallisation method
In order for a protein to crystallise, internal changes to a system are required to cause the protein to exceed its limit of solubility, resulting in a transition from a thermodynamically stable state to an unstable supersaturated state. This can be achieved by adjusting the parameters of a system including composition of the aqueous protein solution (precipitants or additives); pH; temperature. It is during supersaturation where the protein can form intermolecular contacts, spontaneously generating either crystal nuclei or amorphous precipitate. Following nucleation, crystal growth can proceed, sequestering protein from the surrounding solution. Vapour diffusion is the conventional method for altering the precipitant concentration and bringing the protein into a supersaturated state. The sitting drop and hanging drop methods of vapour diffusion were used in this work and are explained diagrammatically with the corresponding phase diagram in Figure 2.3.
Figure 2.3: Crystallisation by vapour diffusion. (a) A cross-section of the experimental set-up of vapour diffusion methods. An unsaturated protein solution containing precipitant ‘sitting’ in a well ((i) sitting drop) or suspended over the precipitant reservoir ((ii) hanging drop). The [protein] increases as vapour equilibration between the droplet and reservoir occurs, bringing the protein into a supersaturated state. (b) Phase diagram of vapour diffusion. Vapour diffusion results in the trajectory of the protein solution from an unsaturated to a supersaturated state. Nucleation and crystal growth occur in the supersaturated zone but require distinct conditions: high (labile) and low (metastable) supersaturated states respectively. Nucleation does not occur if the supersaturated state is too low (metastable zone) or too high (precipitation zone, which leads to the formation of precipitate). Following nucleation in the labile zone, the protein will be sequestered as the crystal grows, returning the solution to the metastable zone. The black arrow indicates the ideal strategy for crystal growth.
[P ro te in ] [Precipitant] SUPERSATURATION UNSATURATION METASTABLE LABILE PRECIPITATION ZONE Nucleation/ Growth Growth
(i) Sitting drop (ii) Hanging drop
Precipitant reservoir (100 µL) Clear film seal
Protein-precipitant sample well Precipitant reservoir (1 mL) Grease-sealed coverslip Protein- precipitant droplet
(a)
(b)
2.10.2. Sparse matrix screens
Sparse matrix screens have been developed with a bias towards conditions that have successfully generated protein crystals with an emphasis on either soluble or transmembrane proteins. The screens are used as a starting point when studying a protein that has not been previously crystallised. Soluble protein sparse matrix screens utilised include: Wizard I screen (Emerald Biosciences); Newcastle I screen (University of Newcastle); Clear Strategy I screen (Molecular Dimensions); Index
screen (Hampton Research); JCSG-plus (Molecular Dimensions) and PACT-premier
(Molecular Dimensions). Transmembrane protein sparse matrix screens include: MemStart (Molecular Dimensions), MemSys (Molecular Dimensions) and MemGold (Molecular Dimensions). The Honeybee 963 crystallisation robot (Digilab) was used as an automated facility to set up the crystallisation screens in a sitting-drop format in 96-well Greiner plates or 96-well MRC plates, using a volume of 100 µL precipitant per reservoir. The sitting drops consisted of 200 nL protein mixed with 200 nL precipitant and the trials were carried out at 18°C unless otherwise stated.
2.10.3. Optimisation screening 2.10.3.1. In-house screen development
Following an indication of a successful crystal nucleation and growth in a sparse matrix condition, the reproducibility of the crystals and improvements in crystal quality were pursued by traversing the initial condition across buffer pH, precipitant concentrations, protein concentrations and protein to precipitant ratio. Crystal screens were set-up using the hanging drop method of vapour diffusion in a 24-well plate, with 1 mL precipitant solution in the reservoir and total droplet size of 2 µL (containing protein and precipitant). The trials were carried out at 18°C.
2.10.3.2. Additive screen
The Additive screen (Hampton Research) is a library of small molecules introduced into crystallisation screens to manipulate the conditions. The crystallisation trials
were performed according to the manufacturer’s instructions in a 96-well plate using the sitting drop vapour diffusion method. The trials were carried out at 18°C.
2.10.3.3. Silver Bullet screen
The Silver Bullet screen (Hampton Research) contains small molecules that have been documented to promote crystal lattice formation. The crystallisation trials were performed according to the manufacturer’s instructions using the sitting drop vapour diffusion method in a 96-well plate. The trials were carried out at 18°C.
2.10.4. Cryoprotection
The X-ray exposure to protein crystals can cause secondary radiation damage within the crystal lattice, where free radicals are generated at room temperature (Garman and Doublié, 2003). To minimise this effect and to preserve the life of the crystal, data collection was carried out at 100°K in a nitrogen vapour stream, allowing better quality data to be obtained at a higher resolution. Cryoprotectants were employed during the freezing of the crystal to allow the formation of vitreous ice as opposed to crystalline ice, by reducing the water molecules in contact with each other, thus avoiding disruption of the crystal lattice. In this work, 20-30 % (v/v) glycerol was used as a cryoprotectant. Crystals were mounted with rayon fibre tubes of 0.05-0.3 mm, held within the loop by surface tension. Crystals were transferred to 5 µL drop of mother liquor containing cryoprotectant (replacing the appropriate volume of water). In the cryoprotectant of 30 % (v/v) glycerol, crystals were soaked in a three- step gradient. The loop was plunged into liquid nitrogen to freeze the crystal. Frozen crystals were stored in a Taylor Wharton dewar.
2.10.5. Data collection
Preliminary diffraction data was obtained using the in-house X-ray facility. Complete data sets were collected at the Diamond Lightsource Radiation Facility (UK) on the I24 microfocus beamline using a Pilatus 6M solid state detector with an oscillation angle of 0.2° and a crystal to detector distance of 416 mm.