Crystallisation principles

Dr Rupert C. Wilmouth
School of Biological Sciences
NTU
 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 µL) suspended
above 100 µL – 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.
Hanging drop v. sitting drop

Hanging drop Sitting drop

ƒ Cheaper ƒ Able to use larger drops
ƒ Easier to setup ƒ Easier for soaking
ƒ Easier to harvest crystals ƒ Safer for transport
ƒ Multiple drops possible on one cover slide
Crytallisation kinetics – phase diagrams

Vapour diffusion – no crystals formed

Crytallisation kinetics – phase diagrams

Vapour diffusion – crystals formed

 Microbatch
„ A 1-2 µL 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 µL or below makes the method very
suitable for screening.
Crytallisation kinetics – phase diagrams

Microbatch – constant protein concentration

 Other crystallisation methods
„ Another method used to bring about gradual change in the relative
concentrations of the protein and precipitant is dialysis. This ranges from
traditional dialysis tubing for milliliter samples to the use of specialised
dialysis buttons which make it easy to dialyse as little as 10 µL of sample. The
use of dialysis permits one to increase the concentration of precipitant while
essentially holding protein and other component concentrations steady. It is
also possible to induce crystallisation through a change in pH by dialysing
with a pH gradient.
„ Hydrogels can be used for crystal growth with the crystallisation solution
soaking a polymeric network. Two common examples are agarose and silica.
A silica hydrogel can be formed by reacting sodium silicate with acetic acid.
Gels can reduce nucleation and allow crystals to grow larger. They can also
provide stability to fragile crystals. The main disadvantage is the extra hassle
involved and it can also make crystal manipulation more difficult.
 Sparse matrix screens
„ Sparse matrix screens are a common method for initially
determining potential crystallisation conditions. A carefully chosen
wide selection of buffers, pH values, salts and precipitants is
screened.
„ The most widely used sparse matrix screen is the one originally
sold by Hampton and now copied by Molecular Dimensions and
Sigma-Aldrich (all three are identical).
„ Many other sparse matrix screens have been devised (see the
Molecular Dimensions catalogue), and it is worth trying these if
the ‘Hampton’ screen does not yield interesting results.
„ Remember to make sure that your protein solution is only weakly
buffered, otherwise the pH value of the screen will be
meaningless. Also, ensure that no other salts or additives have
been added to your protein solution via the purification procedure.
Empty drop with some light ‘skin’
Empty drop with light precipitate
Empty drop with heavy precipitate
Heavy precipitate and crystals
Oil droplets – phase separation
Needle cluster
Single needles
Needles from oil droplets
Twinned plates
Rectangular crystals
Hexagonal crystals (lysozyme)
Small thin crystals
Small rectangular crystals
Salt crystals
Dried out PEG droplet
Air bubble
Fungal growth
Crystal optimisation

Dr Rupert C. Wilmouth
School of Biological Sciences
NTU
Optimisation of conditions
„ Grid screens
„ Temperature (e.g. 4°C, 10°C, 18°C)
„ 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?
 [PEG 8000] (w/v)
4% 6% 8% 12% 16% 20%

0.1 M HEPES pH 7.0

0.1 M HEPES pH 7.5

0.1 M TRIS pH 8.0

0.1 M TRIS pH 8.5

 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-β,D-glucoside, NDSB-195 etc.),
chaotropic agents (urea, guanidine), sugars (e.g. sucrose, glucose
etc.), alcohols (e.g. methanol, ethanol, isopropanol, glycerol, 2-
methyl-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).
 Different protein constructs
„ Often it is easier to try a different protein construct
than trying the thousandth different additive in the
hope of improving your crystal quality.
„ Possible changes include moving the location of the
6His tag, deleting the 6His tag (native purification),
using a different tag (e.g. GST), performing N-
terminal or C-terminal truncations (try deleting
residues, one at a time).
„ Other possibilities may include changing the
expression system, fungal, insect or mammalian cells
are alternatives to bacterial expression.
 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 cat’s 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 µL) 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, cat’s
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.
 Cryo-annealing and dehydration
„ These are last ditch attempts to improve the diffraction
of your crystals.
„ Cryo-annealing is where your frozen crystal is (briefly)
defrosted before being refrozen. Occasionally the
mosaicity (and less often the resolution) can
dramatically improve.
„ Dehydration is the intentional drying out of your
crystal. This can reduce the water content of your
crystal, and hence perhaps improve the resolution.
Two possibilities: stepwise increase of your precipitant
concentration, or use a hanging drop suspended above
a humectant solution (e.g. 50% or 75% glycerol).