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Disclaimer
This is my version of a basic-to-advanced guide to the
crystallization of proteins. Actually, it's just excerpts from
Crystallization Theory
Crystallization is one of several means (including nonspecific
aggregation/precipitation) by which a metastable supersaturated
solution can reach a stable lower energy state by reduction of
solute concentration (Weber, 1991). The general processes by
which substances crystallize are similar for molecules of both
microscopic (salts and small organics) and macroscopic (proteins,
DNA, RNA) dimensions. There are three stages of crystallization
common to all systems: nucleation, growth, and cessation of
growth.
Nucleation is the process by which molecules or noncrystalline
aggregates (dimers, trimers, etc.) which are free in solution come
together in such a way as to produce a thermodynamically stable
aggregate with a repeating lattice. Crystallization is known to
lower the free energy of proteins by ~3-6 kcal/mole relative to the
solution state (Drenth and Haas, 1992). The formation of
crystalline aggregates from supersaturated solutions does not
however necessitate the formation of macroscopic crystals.
Instead, the aggregate must first exceed a specific size (the critical
size) defined by the competition of the ratio of the surface area of
the aggregate to its volume (Feher and Kam, 1985; Boistelle and
Crystallization Methodology
Solution properties influencing crystallization Crystallization of
macromolecules is a paradigm on par with protein folding as to the
number of possible substates which exist in excess of the global
minimum where perfect crystals form (Feher and Kam, 1985). The
reasons behind the high degree variability in the crystallization of
macromolecules are manifold and include: 1) the high degree of
mobility at the surface and as a whole for large molecules, 2) the
electrostatic nature of macromolecules (i.e. their composition as a
random assortment of mobile point charges embedded in a flexible
low dielectric medium), 3) the relatively high chemical and
physical instability of macromolecules (unfolding, hydration
requirements, temperature sensitivity), and 4) molecule specific
factors (such as prosthetic groups and ligands). These factors
contribute greatly to the difficulty and crudity in current attempts
to predict crystallization behavior by de novo calculations even for
relatively small and stable proteins like lysozyme (Durbin and
Feher, 1991). The above factors are also reflected in the general
trend towards greater crystal disorder (and lower resolution of
Table 1.1
Effective precipitant salts
Precipitant
Effective Concentration
Max Concentration
-----------------------------------------------------------------------(NH4+/Na+/Li+)2 or Mg2+
generally 50-80%
SO42saturation
NH4+/Na+/K+ PO43generally 50-80%
saturation
NH4+/K+/Na+/Li+ citrate
1.2-1.8 M
NH4+/K+/Na+/Li+ acetate
generally 50-80%
all ~1.8 M
~3.0 M
saturation
NH4+/K+/Na+/Li+ Clgenerally 50-80%
saturation
NH4+NO3generally ~4.0 M
KSCN
~200 mM (?)
~8.0 M
?
precipitants (i.e. salts and PEG's, MPD and salts, salts and
organics, etc.) may produce larger, better diffracting crystals, and
occasionally new space groups. Some of the best combinations
(based on results of crystallization trials) are outlined in Table 1.2.
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Table 1.2
Proven combinations of precipitants
Major Precipitant
[Additive]
Additive
[Major Precipitant]
-----------------------------------------------------------------------(NH4)2SO4
6%-0.5%
PEG 400-2000,
2.0-4.0 M
MPD, ethanol,
methanol
Na citrate
6%-0.5%
PEG 400-2000,
PEG 1000-20000
0.2-0.6 M
(NH4)2SO4, NaCl,
1.4-1.8 M
MPD, ethanol,
methanol
40-50%
or Na formate
------------------------------------------------------------------------
equals that of the well solution. Since the volume of the well
solution is much greater than that of the drop (1-3 ml as compared
1-20 ul) its dilution by the water vapor leaving the drop is
negligible.
Fowlis and coworkers have demonstrated that the rate of vapor
equilibration in normal (1 g) gravity is dependent strictly on the
rate of vapor diffusion of water in the space separating the drop
and the well (Fowlis et al., 1988). Due to convection effects
(caused by the increased concentration of the precipitant at the
edge of the drop as water evaporates), the rate of diffusion of a
water molecule in the suspended drop (in solution) is actually
greater than that of the vaporized water molecule. In microgravity
experiments (zero effective g), the rate of equilibration is based
solely on the rate of diffusion of water in the drop until crystal
nucleation, at which point in time Marangoni convection (due to
concentration gradients or surface tension around the crystal)
increases the rate of equilibration (Provost and Robert, 1991).
Provost and Robert report that the use of simple agarose gels can
offset convection currents under normal gravity producing
improved results with hanging drops.
Vapor diffusion is the optimal technique to use either when
screening a large number of conditions (by varying the
composition of each well solution) or when dearth of protein
prevents the use of other methods. Furthermore, this method can be
used to increase or decrease the concentration of protein in the
equilibrated state relative to its initial concentration. This is done
by varying the volume of protein mixed with the well solution
when the drop is initially setup. Since the drop equilibrates so that
the precipitant concentration matches that of the well solution, the
final volume of the drop will always equal that of the initial
amount of well solution mixed with the protein. Thus, if the
protein solution is mixed in a 2:1 ratio with the well solution, the
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Table 1.3
Additives for perturbation screening
(In addition to the major precipitant, e.g. ammonium sulfate)
Increased
Other
Dielectric
Decreased
Detergents
Dielectric
-----------------------------------------------------------------------200 mM Na/K/LiCl
5-50 mM spermine
0.25-1% BOG
isopropanol, or
tert-butanol
200 mM Na formate
1% 18-6 crown
ether
1% MPD
0.25-1% BNG
200 mM Na2/K2HPO4
5-50 mM CdSO4,
CoSO4, or NiSO4
20-50 mM urea,
1-5 mM DTT,
trichloroacetate,
cysteine or 1-20
guanidium HCl, or
mM AgNO3
KSCN
0.15% 1,2,3
600, or 1000
heptanetriol
0.1-1% dioxane
750, or 2000
pH 8.6,20%
pH 5.0,2.0 M
pH
pH 8.6,35%
pH 5.0,2.5 M
pH
MPD
Na Citrate
Na/K Phosphate
pH 5.8,30%
pH 7.6,30%
pH 5.8,1.3 M pH 7.5,1.3 M
pH 6.0,2.0 M pH 7.4,2.0 M
pH 5.8,50%
pH 7.6,50%
pH 5.8,1.5 M pH 7.5,1.5 M
pH 6.0,2.5 M pH 7.4,2.5 M
-----------------------------------------------------------------------------------------------------------------------------------------------
tray 2
----------------------------------------------------------------------------------------------------------------------------------------------PEG 2000 MME/0.2 M A.S.
for wells 31-48
pH 5.5,25% pH 7.0,25%
pH 8.5,25%
32
33
pH 5.5,40% pH 7.0,40%
pH 8.5,40%
35
36
41
47
37
43
38
42
44
48
Random
31
34
39
40
45
46
----------------------------------------------------------------------------------------------------------------------------------------------A cheap, fast, and effective initial screen The above screen is an
integration of the footprint screen of Stura with a grid approach,
plus some sparse conditions thrown in as well. It has been effective
Seeding
Seeding is a method of insuring or triggering (in the case of
heterogeneous nucleation) nucleation and crystal growth (Stura
and Wilson, 1990, Thaller et al., 1981; McPherson, 1988).
Commonly a protein crystal or crystalline aggregate is placed in a
storage solution and then crushed to a fine powder (producing a
large number of microscopic crystals) and mixed to make a seed
solution. The seed solution is then added to the supersaturated
crystallization trial (e.g. added to the equilibrated hanging drop or
batch tube) thus ensuring the presence of nuclei for crystal growth.
If no crystalline material is available for a particular protein, seed
solutions from related proteins (e.g. the same protein, but from a
different species) may sometimes trigger crystallization of the
target protein.
Microanalytical seeding This technique is designed to analyze the
results of crystallization trials in which some or all of the
conditions failed to yield crystals. It is best done with hanging or
sitting drops but may be done with other methods as well. In short,
high concentrations of seeds are added to all trials and the results
are judged based on the morphology of the resultant crystals. In
References
Althoff, S. M. The crystallization and preliminary X-ray analysis
of Escherichia coli adenylate kinase. Bachelors Thesis. University
of Illinois at Urbana. 1987.
Althoff, S., Zambrowicz, B., Liang, P., Glaser, M., and Phillips, G.
N. Jr. Crystallization and preliminary X-ray analysis of Escherichia
coli adenylate kinase. J. Mol. Biol. 199:665-666, 1988.
Arakawa, T. and Timasheff, S. N. Theory of protein solubility.
Methods in Enz. 114:49-77, 1985.
Bergfors, T. The crystallization lab manual. 1993.
Berry, M. B., Meador, B., Bilderback, T., Liang, P., Glaser, M.,
and Phillips, G. N., Jr. The closed conformation of a highly
flexible protein: the structure of E. coli adenylate kinase with
bound AMP and AMPPNP. Proteins: Structure, Function and
Genetics. 19:183-198, 1994.
Boistelle, R. and Astier, J. P. Crystallization mechanisms in
solution. J. of Cryst. Growth. 90:14-30, 1988.
Carter, C. W. Jr. and Carter, C. W. Protein crystallization using
incomplete factorial experiments. J. Biol. Chem. 254:1221912223, 1979.
Carter, C. W. Jr., Baldwin, E. T., and Frick, L. Statistical design of
experiments for protein crystal growth and the use of a
precrystallization assay. J. of Cryst. Growth. 90:60-73, 1988.
Conroy, M. J. and Lovrien, R. E. Matrix coprecipitating and
cocrystallizing ligands (MCC ligands) for bioseparations. J. of
Cryst. Growth. 122:213-222, 1992.