Crystallization for class notes

1. Crystallization

2. Four major steps in crystallization
Obtain large amounts of pure protein samples
Choose a protein buffer in which the protein is both
soluble and stable
Bring protein solution to supersaturation where
spontaneous nucleation can take place

3. Solubility
As a rule, protein solubility will usually increase as you add
salt to your aqueous solution, then begin to decrease when the
salt concentration gets high enough to compete with the protein
for hydration (interaction with water molecules).
Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand
http://www2.vuw.ac.nz/staff/alan_clark/teaching/index.htm
HbCO
(carboxyhemoglobin)
solubility as a function
of ionic strength in the
presence of several
different types of salts

4. Nucleation
A phenomenon whereby a “nucleus”, such as a dust particle, a
tiny seed crystal, or commonly in protein crystallography, a
small protein aggregate, starts a crystallization process.
Nucleation poses a large energy barrier, which is easier to
overcome at a higher level of supersaturation.
Common difficulties:
1. If supersaturation is too high, too many nuclei form, hence
an overabundance of tiny crystals.
2. In supersaturated solutions that don’t experience
spontaneous nucleation, crystal growth often only occurs in
the presence of added nuclei or “seeds”.

5. Cessation of growth
Caused by the development of growth defects or the
approach of the solution to equilibrium.
Mother liquor
The solution in which the crystal exists - this is often
not the same as the original crystallization screening
solution, but is instead the solution that exists after
some degree of vapor diffusion, equilibration through
dialysis, or evaporation.

6. Major factors that affect crystallization
1) Purity of proteins
2) Protein concentration
3) Starting conditions (make-up of the protein solution)
4) Precipitating agent (precipitant)
5) Temperature
6) pH
7) Additives: Detergents, reducing agents, substrates, co-factors,
etc.

7. 1) Purity of proteins
Sources of heterogeneity (other than unrelated
proteins and nucleic acids as contaminants):
• Partial proteolysis products
• Oxidation of cysteines
• Deamidation of Asn and Gln to Asp and Glu
• Post-translational modifications
• Oligomerization
• Isoforms
• Misfolded population
• Structural flexibility

8. 2) Protein concentration
Consistency and reproducibility are the major issues
with protein concentration a reliable assay for
determining the concentration.
• Bradford Assay (BSA is used as a standard)
E. Coli expression systems are crystallographers’ most
commonly used method of obtaining protein. Problems
can arise from low expression yields:
• Cytotoxic - your protein is killing your E. coli
• Unstable plasmid or mRNA
• Protein is misfolded (coexpress with GroEL?)
• Some common eukaryotic codons are rare in E. coli

9. 3) Starting conditions (make-up of
the protein solution)
The main point is to KNOW what your starting
conditions are for purposes of reproducibility.

10. 4) Precipitating agent (precipitant)
Salts
Ammonium sulfate
Sodium chloride
Potassium phosphate
Organic reagents
MPD
Isopropanol
Polyethylene glycol
PEG 4000
PEG 6000
PEG 8000

11. Comparison of CrystallizationComparison of Crystallization
and Precipitationand Precipitation
Description Crystallization Precipitation
Solubility Wide range, usually
medium to high
Sparingly soluble
Relative
supersaturation
Low High
Product morphology Well-defined Ill-defined
Product crystal size Large Small
Nucleation
mechanism
Secondary Primary
Nucleation rate Low High
Growth Rate Wide Range Low
Controllability Controllable Difficult to control

12. 5) Temperature
Temperature affects protein stability and also the dynamics of
how protein solution reaching supersaturated states.
Ideally:
• An individual crystal screen should be kept at constant temperature
• Each set of conditions should be screened at several temperatures
• The easiest are 4° C and room temperature, also try 12 or 15° C

13. 6) pH
Surface charges affect “crystal packing”.
(Crystal packing refers to the spatial arrangement of
molecules within the crystal, particularly in
reference to their relationships to one another.)
Hydrophobic interactions are less important than
electrostatic interactions in crystal packing.

14. 7) Additives:
Sometimes you can increase the stability of your protein,
and/or the homogeneity of its conformation by having
relevant additives present in the crystal screen:
• Detergents
• Reducing agents
• Substrates
• Co-factors
• etc.

15. Still no crystals after thorough screening.
Now what?
 New constructs
Deletion mutants
Complexes with substrates
Protein complex with Fab fragments
Homologous proteins
Fab

16. Crystallization of membrane proteins
The lipidic cubic phase method (Landau and
Rosenbusch)
Cocrystallization with Fab fragments

17. Common Methods for Crystallization:
•Vapor Diffusion
•Slow Evaporation
•Dialysis

18. Hanging Drop Vapor Diffusion
Most popular method among
protein crystallographers.
1. Crystal screen buffer is the
well solution (0.5 - 1 mL)
2. Drop (on siliconized glass
cover slip) is 1/2 protein
solution, 1/2 crystal screen
buffer (6-10 µL). So, the
concentration of precipitant in
the drop is 1/2 the
concentration in the well.
3. Cover slip is inverted over
the top of the well and sealed
with vacuum grease (airtight).
4. The precipitant concentration in the drop will equilibrate with
the precipitant concentration in the well via vapor diffusion.

19. Sitting Drop Vapor Diffusion
Same basic principles
as in hanging drop
method, except the drop
containing your sample
sits on a bridge within
the well. This allows for
a larger sample size (20 -
40 µL), however protein
is frequently precious to
the crystallographer, so
there isn’t that much
demand for a larger sample
size.

20. Oil Immersion Micro Batch
This method is rising rapidly in
popularity- typical sample size 1-6 µL
Figure 1- Paraffin oil allows for little to no
diffusion of water through the oil. This is a
true batch experiment because all the
reagents are present at a specific and
relatively unchanging concentration.
Figure 2- Al’s oil is a 1:1 mixture of
silicon oil and paraffin oil which allows
for evaporation through slow diffusion
through the oil. This is an evaporation
Method, and the concentration of the
protein and reagents in the drop does
increase over time.

21. Microdialysis
Dialysis buttons can be
purchased for a wide range
ofsample sizes (~ 5 - 350 µL).
In the dialysis experiment, the
sample is often introduced to
high salt concentrations within
the button that are allowed to
equilibrate with lower salt
concentrations in the buffer
over time. This is known as a
“salting-in” method. It
exploits the fact that not only
does protein solubility tend to
decrease with very high ionic
strengths, it also has a
minimum at very low ionic
strength.

22. Nucleation
The generation of ultramicroscopic particles in the process of nucleation is
the sum of contributions by primary nucleation and second nucleation.
Primary nucleation : occurs in the absence of crystals, secondary
nucleation: attributed to the influence of existing crystals
Primary nucleation can be either homogeneous (no foreign particles are
present) or heterogeneous (foreign particles present during heterogeneous
nucleation)
Rate of primary nucleation has been modeled by the following power law
expression:
Crystallization PrinciplesCrystallization Principles
B: number of nuclei formed per unit volume per unit
time;
N: number of nuclei per unit volume; kn : rate constant;
c: instantaneous solute concentration; c*: solute
concentration at saturation. (c-c*) term :
supersaturation,
the exponent of n can range up to 10 but typically is in
the range of 3 to 4.
B: number of nuclei formed per unit volume per unit
time;
N: number of nuclei per unit volume; kn : rate constant;
c: instantaneous solute concentration; c*: solute
concentration at saturation. (c-c*) term :
supersaturation,
the exponent of n can range up to 10 but typically is in
the range of 3 to 4.

23. CrystallizationCrystallization
PrinciplesPrinciples
Two types of secondary nucleation : shear nucleation
(occurs as a result of fluid shear on growing crystal faces),
contact nucleation ( happens because of crystals colliding
with each other and with the impeller and other vessel
internal surfaces.
Rate of secondary nucleation in crystallization is the
following:
(2)
k1 : rate constant; MT : suspension density, b : can range up to
5 but has a most probable value of 2; j: ranges up to 1.5
with 1 being the most probable value
k1 : rate constant; MT : suspension density, b : can range up to
5 but has a most probable value of 2; j: ranges up to 1.5
with 1 being the most probable value

24. Crystallization PrinciplesCrystallization Principles
Figure 1: Typical phase diagram. The components in
solution consist of the product (ordinate) and the
precipitating reagent (abscissa). The lines with arrows
out line one possible way of performing the
crystallization.
- The supersaturation must be above the a
certain value before nucleation will begin
- Metastable region : the supersaturation is low
that nucleation will not start
- Once the supersaturation has been raised
enough to be in the labile region, nucleation
can begin.
- At this point, crystals begin to grow, and the
supersaturation decreases
- If the supersaturation becomes too high, the
nucleation rate will be too great, and
amorphous precipitate will result.

25. Crystallization PrinciplesCrystallization PrinciplesNucleation

26. SupersaturationSupersaturation
Precipitatant concentration (salt, PEG etc.)
Proteinconcentration
Under-saturation
(protein remains soluble; crystals dissolve)
Nucleation zoneNucleation zone
Precipitation zonePrecipitation zone
Solubility
curve
Metastable zoneMetastable zone
Crystals grow, butCrystals grow, but
Nuclei form onlyNuclei form only
infinitely slowlyinfinitely slowly

27. [Precipitatant]
Proteinconcentration
NucleationNucleation
PrecipitationPrecipitation
MetastableMetastable
Start w/
soluble protein
(undersaturated
or metastable)
NucleatesNucleates
herehere
Increase
[protein],
[precipitant]
Crystal growsCrystal grows
Sequesters proteinSequesters protein
[protein] drops[protein] drops
Crystal stops growing @Crystal stops growing @
solubility curvesolubility curve
Expt incr. [protein], [precipitant]
Xtl grows again, until hits curve
Repeats as follows solubility curve

28. Crystal Growth
Post nucleation process in which molecules in solution are added
to the surface of existing crystals
The rate of mass deposition R during crystal growth is:
Overall linear growth rate can also be expressed as:
L : characteristics single dimension of the crystal, such as length
Crystallization PrinciplesCrystallization Principles
(3)
(4)
W: mass of crystals per volume of solvent;
A : the surface area of crystals per volume of
solvent;
kG : overall mass transfer coefficient (depends on
temperature, crystal size, hydrodynamic
conditions, the presence of impurities);
g : usually 0 and 2.5
W: mass of crystals per volume of solvent;
A : the surface area of crystals per volume of
solvent;
kG : overall mass transfer coefficient (depends on
temperature, crystal size, hydrodynamic
conditions, the presence of impurities);
g : usually 0 and 2.5

29. CrystallizationCrystallization
PrinciplesPrinciples
Crystal growth is a process that consists of two
steps in series – diffusion and surface integration
When the exponents are unity, combining Equation 3, 5,
6 gives
(5)
ci : concentration at the interface between the liquid
and solid phase; kd and kr : mass transfer
coefficients
(6)
(7)
Thus, if surface integration is very fast
compared with bulk diffusion, then kr >> kd,
and kG , kd.

30. Yields and Heat and MaterialYields and Heat and Material
Balances in CrystallizationBalances in Crystallization
Yields and material balance in crystallization
The solution (mother liquor) and the solid crystals are in
contact for enough time to reach equilibrium. Hence, the mother
liquor is saturated at the final temperature at the final temperature
of the process, and the final process, and the final concentration of
the solute in the solution can be obtained from the solubility curve.
The yield can be calculated knowing the initial concentration
of solute, the final temperature, and the solubility at
this temperature.
In making the material balances, the calculations are
straightforward when the solute crystals are anhydrous. Simple
water and solute material balances are made. When the
crystallizations are hydrated, some of the water in solution is
removed with the crystals as a hydrate.

31. Properties of protein crystals
Soft, easy to crush
Contain large solvent channels
Relatively large organic and inorganic molecules can
diffuse inside
Anisotropic physical properties
Birefrigence due to anisotropic refraction indices
Ability to diffract X-ray due to regular spaced
lattices

32. New Techniques: Recrystallization
Recrystallization: method of purifying an organic solid
Gravity Filtration: method of removing insoluble
impurities from recrystallization solution
Suction Filtration: method of isolating pure solid from liquid
(filtrate) using vacuum

33. In Lab
Recrystallization
Determine best solvent for recrystallization
Weigh crude solid
Recrystallize
Weigh purified product

34. After Lab
Let recrystallized solid air dry on shelf.
Weigh dry crystals and take a melting point (next week).
Calculations:
% recovery:
[mass crude/mass recrystallized] x 100
Conclusions: Discuss effectiveness of recrystallization process
in terms of purity and percent recovery. Discuss role/attributes
of solvent, sources of error, etc.

35. Pure solid: Tight
crystal lattice Impurities disrupt the
crystal lattice

36. Steps in Recrystallization1. Dissolve crude solid in hot solvent (saturated solution)
2. Let solution cool to room temperature, so that crystal
lattice reforms.
3. Cool solution in an ice bath.
4. Suction filter the pure solid, leaving impurities in
solution.
5. Let solid air dry to remove traces of solvent.

37. 4. Suction filter solid away from
impurities.
5. Let crystals air dry.

38. An Ideal Recrystallization Solvent Should
should dissolve all of the compound when the solvent is hot
(boiling).
should dissolve none of the compound when the solvent is
at room temperature.
should have different solubilities for the compound and the
impurities.
should have a lower boiling point than the melting point of
the compound.
should have a fairly low boiling point
should be cheap, non-toxic, non-reactive, and non-smelly

39. Crystallization
Nucleation
Crystallization and Nucleation