Professional Documents
Culture Documents
Crystallization
Crystallization
Protein crystals
Proteins are inherently hard to crystallise and protein crystals are fragile compared to crystals of inorganic compounds. Protein crystals are held together by weak forces, primarily hydrogen bonds. Crystals only diffract X-rays when wet. Initial experiments in the early 20th century using dried protein crystals gave no diffraction. Finally, in 1934, Bernal and Hodgkin measured diffraction from pepsin crystals that were still in their mother liquor, i.e. wet.
Growing crystals
In order to grow protein crystals, the protein must be pure (usually >95%), homogeneous, and in high concentration (> 5 mg mL-1). Since pH greatly affects crystallisation, a buffer must be present (e.g. MES, HEPES, TRIS). Finally, a precipitant is also required which causes the protein to precipitate out of solution. Under careful controlled conditions (especially temperature and evaporation rate), protein solutions will sometimes become supersaturated instead of precipitating. At this critical point, crystals may form. Nucleation, the formation of the first ordered aggregates, is a key event. Nucleation conditions are sometimes difficult to reproduce, and thus seeding procedures with preformed crystalline material may be invaluable to obtain reproducible results.
Vapour diffusion
A drop of protein solution is mixed with an approximately equal amount of crystallisation solution from a reservoir. The reservoir or well has a greased rim and is then sealed with a cover slide (either siliconized glass or plastic). In the resulting closed system, water vapour diffuses from the hanging drop into the reservoir, which contains about twice the precipitant concentration than the drop. This allows the protein to become more concentrated and potentially allows crystallisation to occur. The drop can either be placed on the underside of the cover slide (hanging drop) or placed on a plastic support above the surface of the reservoir (sitting drop). The method can be used for relatively large drops (2-20 L) suspended above 100 L 1 mL of reservoir solution in 24-well plates. It can also be used for micro-drops in small crystallisation strips. The smallest drop size is limited by the evaporation during the time delay from drop application to sealing of the well.
Sitting drop Able to use larger drops Easier for soaking Safer for transport
Microbatch
A 1-2 L drop of protein solution mixed with an aliquot of crystallisation solution (containing precipitant, possible additives, buffers, etc.) is pipetted onto the surface of a oil-covered microtitre plate well. The drop then sinks to the bottom of the well, and is isolated from the environment. Variations include placing the individual components under oil, with either protein drop or crystallisation solution first (creating two kinetically different scenarios), and using varying ratios of water permeable oils to allow water to diffuse into the environment. It should be noted that alcohols, detergents, and lipids can diffuse into the oil (and, to a much smaller degree, into the polymer of the well material). The micro-batch method is well suited for miniaturisation and automation, as there is no time delay between the application of the drop and the sealing. The ability to work with minute drops of 1 L or below makes the method very suitable for screening.
Needle cluster
Single needles
Twinned plates
Rectangular crystals
Salt crystals
Air bubble
Fungal growth
Crystal optimisation
Dr Rupert C. Wilmouth School of Biological Sciences NTU
Optimisation of conditions
Grid screens Temperature (e.g. 4C, 10C, 18C) Additives Cations (anions) Substrates/inhibitors Limited proteolysis Different protein constructs
Grid screens
Grid screens are useful for screening two conditions against each other. Three dimensional grid screens are also possible by using multiple trays. Grid screens take a fair amount of effort to make up, therefore choose your conditions carefully. Also, take extreme care not to make errors one mistake and you may miss the correct conditions. In general, screen precipitant conditions more widely than pH. Most proteins crystallise between pH 6 and pH 8.5. Make sure you use the correct buffer (i.e. within 0.5 pH of the pKa). For example, you find some promising looking precipitate in Hampton condition 36 (0.1 M TRIS pH 8.5, 8% (w/v) PEG 8000). What grid screen might one use next?
Additives
There are a huge number of additives that can be tried. Usually this is the last thing to be screened. Additives are most useful when your crystallisation conditions are almost correct, for example, when you want to change the morphology of your crystals or increase their size. Examples include cations (e.g. Ca, Fe, Cd, Zn, Ni etc.), detergents (e.g. n-octyl-,D-glucoside, NDSB-195 etc.), chaotropic agents (urea, guanidine), sugars (e.g. sucrose, glucose etc.), alcohols (e.g. methanol, ethanol, isopropanol, glycerol, 2methyl-2,4-pentanediol etc.).
Substrates/inhibitors
If there are substrates, substrate analogs or inhibitors available, then co-crystallisation should always be attempted. The presence of a molecule bound in the active site often makes the protein less flexible and hence easier to crystallise. When you solve the structure, having a substrate or inhibitor bound in the active can give immensely useful mechanistic information. Be careful when using substrates to ensure they are not turned over by the enzyme. You can use non-hydrolysable substrate analogs (e.g. ADPNP instead of ATP) or inactive conditions (e.g. low pH).
Seeding
A seed provides a template for the assembly of molecules to form a crystal with the same characteristics as the crystal from which it originated. It is important to differentiate the process of crystal growth from nucleation. In general, the degree of supersaturation required for nucleation is higher than that required for crystal growth. Normally, during aggregation, there is an equilibrium between the formation of ordered nuclei (reversible) and the formation of precipitate (irreversible). When crystal seeds are added, the equilibrium can shift towards crystal formation, and avoids the random nature of spontaneous nucleation.
Seeding
There are three commonly used types of seeding: Microseeding adding very small pieces of crushed protein crystals to the crystallisation drop. Macroseeding adding an intact, already grown crystal to the crystallisation drop. Streak seeding similar to microseeding but uses a fine hair (often a cats whisker is best) to pick up small protein crystal fragments.
Microseeding
This is generally the easiest and most reliable form of seeding. A few small crystals, or one large crystal, is broken up to a fine powder using a needle. A small amount of mother liquor containing this fine powder is added to an aliquot of well solution and vortexed well. Serial dilutions of the microseed solution can then be carried out (e.g. 1:10, 1:100, 1:1000). Finally, a small quantity (e.g. 0.5 or 1 L) of the microseed solution is added to the protein crystallisation drop. Often the microseed solution is added at the same time that the crystallisation drop is set up. However, it is important to remember that adding the microseed solution to an already equilibrated drop can be advantageous.
Streak seeding
This is a quick method of seeding, and can be used to seed a series of drops rapidly. The main difficulty is to find a suitable fibre to use, cats whiskers are excellent (if you can find a willing cat!). For ease of use, superglue the whisker onto a plastic rod. Degrease the whisker with ethanol or methanol, and then rinse with distilled water. Either, touch an existing crystal with the fibre to dislodge a few seeds, or stroke the fibre through a microcrystalline precipitate. Then introduce the seeds into a fresh drop by stroking the fibre in a straight line through the drop.
Macroseeding
This is perhaps the most difficult seeding technique to use successfully. A single crystal, free from twinning or other crystalline deformities, is selected. This crystal is then washed in a slightly dissolving solution to remove the top layer which may contain invisible defects. The solution must not cause excessive etching or cracking. After washing several times, the crystal is then transferred to fresh drop, preferably pre-equilibrated. Extreme care has to be taken when handling the crystal to avoid damage and the generation of microseeds. Needles are particularly difficult to handle.
Cryo-conditions
These days, mounting of most protein crystals on an X-ray diffractometer is done at cryogenic temperatures, usually around 100K. A small nylon loop is used to pick up a crystal, immerse it a cryoprotectant solution for a short period of time, and then either plunge it in liquid nitrogen (or sometimes liquid propane), or place it directly on the goniometer head in a stream of cold nitrogen gas. The choice of cryoprotectant is critical. It must prevent ice formation and it must not damage the crystal. Always screen several cryoprotectants (e.g. glycerol, ethylene glycol, MPD, PEG 400, sucrose etc.) at several different concentrations, and at several different soaking durations. Do not forget the possibility of mounting your crystal at room temperature in a quartz capillary. This is a very difficult technique to master, but remains the best way of checking the diffraction of your crystals.